Substituted pyrazino[1&#39;,2&#39;:1,5]pyrrolo[2,3-b]indole-1,4-diones for cancer treatment

ABSTRACT

The synthesis of various pyrazino[1′,2′:1,5]pyrrolo[2,3-b]-indole-1,4-dione analogs has been successfully implemented in the present application. From these efforts, compounds having the structure of Formula I-c: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 1 , R 2 , R 4 -R 8 , R 3′ , R 6′ , and n are as defined herein, are provided. These biologically active derivatives have been further used to prepare cell-specific drug conjugates effective in treating various diseases including cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/150,786, filed May 10, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/096,158, filed Dec. 4, 2013; which claimspriority to U.S. Provisional Application Nos. 61/733,222, filed Dec. 4,2012; 61/823,714, filed May 15, 2013; and 61/868,173, filed Aug. 21,2013, the entirety of each of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. GM089732awarded by the National institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to, among other things,compounds, compositions and methods for treating diseases, for example,various cancers.

BACKGROUND

Epipolythiodiketopiperazine (ETP) alkaloids display a broad spectrum ofbiological activities ((a) T. W. Jordan and S. J. Cordiner, TrendsPharmacol. Sci., 1987, 8, 144; (b) C.-S. Jiang and Y.-W. Guo, Mini Rev.Med. Chem., 2011, 11, 728), including antibacterial (C.-J. Zheng, C.-J.Kim, K. S. Bae, Y.-H. Kim and W.-G. Kim, J. Nat. Prod., 2006, 69, 1816),anticancer ((a) P. Waring and J. Beaver, Gen. Pharmac., 1996, 27, 1311;(b) A. L. Kung, S. D. Zabludoff, D. S. France, S. J. Freedman, E. A.Tanner, A. Vieira, S. Cornell-Kennon, J. Lee, B. Wang, J. Wang, K.Memmert, H.-U. Naegeli, F. Petersen, M. J. Eck, K. W. Bair, A. W. Woodand D. M. Livingston, Cancer Cell, 2004, 6, 33; (c) D. M. Vigushin, N.Mirsaidi, G. Brooke, C. Sun, P. Pace, L. Inman, C. J. Moody and R. C.Coombes, Med. Oncol., 2004, 21, 21; (d) D. Greiner, T. Bonaldi, R.Eskeland, E. Roemer and A. Imhof, Nat. Chem. Biol., 2005, 1, 143; (e) M.Yanagihara, N. Sasaki-Takahashi, T. Sugahara. S. Yamamoto, M. Shinomi,I. Yamashita, M. Hayashida, B. Yamanoha, A. Numata, T. Yamori and T.Andoh, Cancer Sci., 2005, 96, 816; (f) C. R. Isham, J. D. Tibodeau, W.Jin, R. Xu, M. M. Timm and K. C. Bible, Blood, 2007, 109, 2579; (g) Y.Chen, H. Guo, Z. Du, X.-Z. Liu, Y. Che and X. Ye, Cell Prolif., 2009,42, 838; (h) Y.-M. Lee, J.-H. Lim, H. Yoon, Y.-S. Chun and J.-W. Park,Hepatology, 2011, 53, 171; (i) F. Liu, Q. Liu, D. Yang, W. B. Bollag, K.Robertson, P. Wu and K. Liu, Cancer Res., 2011, 71, 6807; (j) K. Yano,M. Horinaka, T. Yoshida, T. Yasuda, H. Taniguchi, A. E. Goda, M. Wakada,S. Yoshikawa, T. Nakamura, A. Kawauchi, T. Miki and T. Sakai, Int. J.Oncol., 2011, 38, 365; (k) N. Zhang, Y. Chen, R. Jiang, E. Li, X. Chen,Z. Xi, Y. Guo, X. Liu, Y. Zhou, Y. Che and X. Jiang, Autophagy, 2011, 7,598; (1) H. Chaib, A. Nebbioso, T. Prebet, R. Castellano, S. Garbit, A.Restouin, N. Vey, L. Altucci and Y. Collette, Leukemia, 2012, 26, 662;(m) C. R. Isham, J. D. Tibodeau, A. R. Bossou, J. R. Merchan and K. C.Bible, Br. J. Cancer, 2012, 106, 314; (n) M. Takahashi, Y. Takemoto, T.Shimazu, H. Kawasaki, M. Tachibana, Y. Shinkai, M. Takagi, K. Shin-ya,Y. Igarashi, A. Ito and M. Yoshida, J. Antiobiot., 2012, 65, 263; (o)C.-S. Jiang and Y.-W. Guo, Mini Rev. Med. Chem., 2011, 11, 728),antiviral ((a) W. A. Rightsel, H. G. Schneider, B. J. Sloan, P. R. Graf,F. A. Miller, Q. R. Bartz, J. Ehrlich and G. J. Dixon, Nature, 1964,204, 1333; P. A. Miller, K. P. Milstrey and P. W. Trown, Science, 1968,159, 431), antiparasitic, antifungal ((a) J. J. Coleman, S. Ghosh, I.Okoli and E. Mylonakis, PLoS ONE, 2011, 6, e25321; (b) C. Speth, C.Kupfahl, K. Pfaller, M. Hagleitner, M. Deutinger, R. Würzner, I.Mohsenipour, C. Lass-Flörl and G. Rambach, Mol. Immunol., 2011, 48,2122), antimalarial, immunosuppressive, immunomodulatory ((a) A.Müllbacher, P. Waring, U. Tiwari-Palni and R. D. Eichner, Molec.Immunol., 1986, 23, 231 (b) H. L. Pahl, B. Krauss, K. Schulze-Osthoff,T. Decker, E. B.-M. Traenckner, M. Vogt, C. Myers, T. Parks, P. Waring,A. Mühlbacher, A. P. Czernilofsky and P. A. Baeuerle, J. Exp. Med.,1996, 183, 1829; (c) S. Nishida, L. S. Yoshida, T. Shimoyama, H. Nunoi,T. Kobayashi and S. Tsunawaki, Infect. Immun., 2005, 73, 235; (d) P.Waring, R. D. Eichner and A. Müllbacher, Med. Res. Rev., 1988, 8, 499;(e) P. Waring and J. Beaver, Gen. Pharmac., 1996, 27, 1311), phytotoxic(M. Soledade, C. Pedras, G. Séguin-Swartz and S. R. Abrams, Phytochem.,1990, 29, 777), nematicidal (J.-Y. Dong, H.-P. He, Y.-M. Shen and K.-Q.Zhang, J. Nat. Prod., 2005, 68, 1510), antiplatelet (A. Bertling, S.Niemann, A. Uekötter, W. Fegeler, C. Lass-Flörl, C. von Eiff and B. E.Kehrel, Thromb. Haemost., 2010, 104, 270), and anti-inflammatory effects(E. Iwasa, Y. Hamashima and M. Sodeoka, Isr. J. Chem., 2011, 51, 420).Considerable synthetic efforts have been directed toward the synthesisof the ETP core and ETP-containing naturally occurring alkaloids;however, only a very limited number of compounds are accessible in verysmall amounts (P. Waring, R. D. Eichner and A. Müllbacher, Med. Res.Rev., 1988, 8, 499; E. Iwasa, Y. Hamashima and M. Sodeoka, Isr. J.Chem., 2011, 51, 420; for approaches to epipolythiodiketopiperazines,see: (a) P. W. Trown, Biochem. Biophys. Res. Commun., 1968, 33, 402; (b)T. Hino and T. Sato, Tetrahedron Lett., 1971, 12, 3127; (c) H. Poiseland U. Schmidt, Chem. Ber., 1971, 104, 1714; (d) H. Poisel and U.Schmidt, Chem. Ber., 1972, 105, 625; (e) E. Öhler, F. Tataruch and U.Schmidt, Chem. Ber., 1973, 106, 396; (f) H. C. J. Ottenheijm, J. D. M.Herscheid, G. P. C. Kerkhoffand T. F. Spande, J. Org. Chem., 1976, 41,3433; (g) D. L. Coffen, D. A. Katonak, N. R. Nelson and F. D. Sancilio,J. Org. Chem., 1977, 42, 948; (h) J. D. M. Herscheid, R. J. F. Nivard,M. W. Tijhuis, H. P. H. Scholten and H. C. J. Ottenheijm, J. Org. Chem.,1980, 45, 1885; (i) R. M. Williams, R. W. Armstrong, L. K. Maruyama,J.-S. Dung and O. P. Anderson, J. Am. Chem. Soc., 1985, 107, 3246; (j)C. J. Moody, A. M. Z. Slawin and D. Willows, Org. Biomol. Chem., 2003,1, 2716; (k) A. E. Aliev, S. T. Hilton, W. B. Motherwell and D. L.Selwood, Tetrahedron Lett., 2006, 47, 2387; (l) L. E. Overman and T.Sato, Org. Lett., 2007, 9, 5267; (m) N. W. Polaske, R. Dubey, G. S.Nichol and B. Olenyuk, Tetrahedron: Asym., 2009, 20, 2742; (n) B. M.Ruff, S. Zhong, M. Nieger and S. Bräse, Org. Biomol. Chem., 2012, 10,935; (o) K. C. Nicolaou, D. Giguère, S. Totokotsopoulos and Y.-P. Sun,Angew. Chem. Int. Ed., 2012, 51, 728; for selectedepidithiodiketopiperazine total syntheses, see: (a) Y. Kishi, T.Fukuyama and S. Nakatsuka, J. Am. Chem. Soc., 1973, 95, 6492; (b) Y.Kishi, S. Nakatsuka, T. Fukuyama and M. Havel, J. Am. Chem. Soc., 1973,95, 6493; (c) T. Fukuyama and Y. Kishi, J. Am. Chem. Soc., 1976, 98,6723; (d) R. M. Williams and W. H. Rastetter, J. Org. Chem., 1980, 45,2625; (e) G. F. Miknis and R. M. Williams, J. Am. Chem. Soc., 1993, 115,536; (f) E. Iwasa, Y. Hamashima, S. Fujishiro, E. Higuchi, A. Ito, M.Yoshida and M. Sodeoka, J. Am. Chem. Soc., 2010, 132, 4078; (g) J. E.DeLorbe, S. Y. Jabri, S. M. Mennen, L. E. Overman and F.-L. Zhang, J.Am. Chem. Soc., 2011, 133, 6549; (h) K. C. Nicolaou, S. Totokotsopoulos,D. Giguère, Y.-P. Sun and D. Sarlah, J. Am. Chem. Soc., 2011, 133, 8150;(i) J. A. Codelli, A. L. A. Puchlopek and S. E. Reisman, J. Am. Chem.Soc., 2012, 134, 1930; for our synthetic strategies relevant toepipolythiodiketopiperazines, see: (a) J. Kim, J. A. Ashenhurst and M.Movassaghi, Science, 2009, 324, 238; (b) J. Kim and M. Movassaghi, J.Am. Chem. Soc., 2010, 132, 14376).

SUMMARY

Among other things, the present invention recognizes the need forbiologically active compounds for treating various diseases. In someembodiments, the present invention provides a compound having thestructure of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention provides a compound havingthe structure of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention provides drug-ligandconjugate compounds. In some embodiments, the present invention providesa compound having the structure of formula I:

MLD)s]_(t)   II

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention provides a compound havingthe structure of formula II-a:

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention provides a compound havingthe structure of formula II-b:

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention provides a compound havingthe structure of formula III:

H-LD)s   II-b

or a pharmaceutically acceptable salt thereof, wherein the variables aredescribed in detail, infra.

In some embodiments, the present invention recognizes the challenges forpreparing ETP or thiodiketopiperazine alkaloids, or derivatives oranalogs thereof. In some embodiments, the present invention provides amethod for preparing ETP or thiodiketopiperazine alkaloids or aderivative thereof. In some embodiments, the present invention providesa method for preparing a provide compound. In some embodiments, thepresent invention provides new reagents for preparing ETP orthiodiketopiperazine alkaloids or derivatives or analogs thereof. Insome embodiments, the present invention provides new reagents forpreparing a provided compound. In some embodiments, a provided methodand/or reagent provides unexpectedly high synthetic efficiency, forexample, in terms of product yield and/or purity.

In some embodiments, the present invention provides a method for killingor inhibiting proliferation of cells comprising treating the cells withan amount of a provided compound, or a pharmaceutically acceptable saltthereof, being effective to kill or inhibit proliferation of the cells.In some embodiments, the cells are tumor cells or cancer cells. In someembodiments, the present invention provides a method of treating adisease, comprising administering to a subject in need an effectiveamount of a provided compound or a pharmaceutically acceptable saltthereof. In some embodiments, the present invention provides a method oftreating a disease, comprising administering to a subject sufferingtherefrom or susceptible thereto an effective amount of a providedcompound or pharmaceutically salt thereof. In some embodiments, adisease is a cancer, autoimmune disease or infectious disease. In someembodiments, a disease is cancer. In some embodiments, a disease is anautoimmune disease. In some embodiments, a disease is an infectiousdisease. In some embodiments, a provided compound is a compound offormula I-a. In some embodiments, a provided compound is a compound offormula I-b. In some embodiments, a provided compound is a compound offormula II. In some embodiments, a provided compound is a compound offormula III.

In some embodiments, the present invention provides methods of treatingcancer. In some embodiments, the present invention provides a method oftreating cancer in a subject suffering therefrom, comprisingadministering to the subject a therapeutically effective amount of aprovided compound or pharmaceutically acceptable salt thereof. In someembodiments, a provided compound has the structure of formula I-a. Insome embodiments, a provided compound has the structure of formula I-b.In some embodiments, a provided compound has the structure of formulaII. In some embodiments, a provided compound has the structure offormula II-a. In some embodiments, a provided compound has the structureof formula II-b. In some embodiments, a provided compound has thestructure of formula III.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A to 1B. ETP derivatives induce caspase-dependent apoptotic celldeath. FIG. 1A) Analysis of phosphatidylserine exposure and propidiumiodide inclusion at 24 hours in U-937 cells. Compounds were tested at100-times their 72-hour IC₅₀ values [14 (20 nM), 5 (75 nM), 26 (250 nM),and 33 (500 nM)]. STS was used at 50 nM as a positive control forapoptosis. FIG. 1B) Western blot analysis of Pro-C3 and PARP-1 cleavageat 24 hours in U-937 cells using β-actin as loading control. Compoundswere tested as above, with the exception of STS (100 nM); C3=caspase-3;ETP=epipolythiodiketopiperazine; FITC=fluorescein isothiocyanate;IC₅₀=half maximal inhibitory concentration; PARP=poly(ADP-ribose)polymerase 1; Pro-C3=procaspase-3; STS=staurosporine.

FIG. 2. Percent hemolysis following treatment with ETPs from Table E1-2.Error bars represent standard error of the mean, n≥3.

FIG. 3. Crystal Structure of 12-Hydroxytryptophan 3,5-Dinitrobenzamide(+)-E2-14 (3 views).

FIG. 4. Crystal Structure of Salicylic Tetracycle E2-23 (3 views).

FIG. 5. Crystal Structure of Tetracyclic Triacetate E2-28 (3 views).

FIG. 6. Crystal Structure of (+)-Bionectin A p-Nitrobenzoate E2-38 (3views).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCertain Embodiments of the Invention

Among other things, the present invention recognizes the need forbiologically active compounds for treating various diseases. In someembodiments, the present invention provides a compound having thestructure of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   is a single bond or a double bond, as valency permits:-   R¹ is R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R, —S(O)₂OR, —C(R)₂OR, or    —S(O)₂N(R)₂;-   each R is independently hydrogen or an optionally substituted group    selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7    membered saturated or partially unsaturated carbocyclic ring, an    8-14 membered bicyclic or polycyclic saturated, partially    unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 3-7 membered saturated or partially unsaturated    heterocyclic ring having 1-3 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic    saturated or partially unsaturated heterocyclic ring having 1-5    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    or an 8-14 membered bicyclic or polycyclic heteroaryl ring having    1-5 heteroatoms independently selected from nitrogen, oxygen, or    sulfur; or:    -   two R groups are optionally taken together with their        intervening atoms to form an optionally substituted 3-14        membered, saturated, partially unsaturated, or aryl ring having,        in addition to the intervening atoms, 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,-   R² is R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂, —[C(R)₂]_(q)—SR,    —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R, —[C(R)₂]_(q)—OC(O)OR,    —[C(R)₂]_(q)—OC(O)N(R)₂, —[C(R)₂]_(q)—OC(O)N(R)—SO₂R or    —[C(R)₂]_(q)—OP(OR)₂; or    -   R¹ and R² are taken together with their intervening atoms to        form an optionally substituted 4-7 membered heterocyclic ring        having, in addition to the nitrogen atom to which R¹ is        attached, 0-2 heteroatoms independently selected from oxygen,        nitrogen or sulfur;-   each q is independently 0, 1, 2, 3, or 4;-   R³ is an electron-withdrawing group;-   R⁴ is absent when    is a double bond or is R or halogen;-   R⁵ is absent when    is a double bond or is hydrogen or an optionally substituted C₁₋₆    aliphatic group;-   each of R⁶ and R^(6′) is independently R, halogen, —CN, —NO₂, —OR,    —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    or —OSi(R)₃; or    -   R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;-   n is 0, 1, 2, 3, or 4;-   each R⁷ is independently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR,    —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    —P(R)₂, —P(OR)₂, —P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂,    —B(OR)₂, or —Si(R)₃; or:    -   two R⁷ are taken together with their intervening atoms to form        an optionally substituted 4-7 membered ring having 0-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   R⁸ is —(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR, —C(O)R,    —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; and-   R⁹ is —(S)_(p)—R^(y) wherein p is 1-3 such that m+p is 2-4 and R^(y)    is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or    —S(O)₂N(R)₂; or    -   R⁸ and R⁹ are taken together to form —S—,        —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,        —(S)_(m)—C(O)S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,        —(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)—.

In some embodiments, the present invention provides a compound havingthe structure of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein:each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R,—S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂;

-   each R is independently hydrogen or an optionally substituted group    selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7    membered saturated or partially unsaturated carbocyclic ring, an    8-14 membered bicyclic or polycyclic saturated, partially    unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 3-7 membered saturated or partially unsaturated    heterocyclic ring having 1-3 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic    saturated or partially unsaturated heterocyclic ring having 1-5    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    or an 8-14 membered bicyclic or polycyclic heteroaryl ring having    1-5 heteroatoms independently selected from nitrogen, oxygen, or    sulfur; or:    -   two R groups are optionally taken together with their        intervening atoms to form an optionally substituted 3-14        membered, saturated, partially unsaturated, or aryl ring having,        in addition to the intervening atoms, 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,-   each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,    —[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,    —[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,    —[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or    -   R¹ and R² are taken together with their intervening atoms to        form an optionally substituted 4-7 membered heterocyclic ring        having, in addition to the nitrogen atom to which R¹ is        attached, 0-2 heteroatoms independently selected from oxygen,        nitrogen or sulfur;-   each q is independently 0, 1, 2, 3, or 4;-   each R³ is independently an electron-withdrawing group;-   each R⁵ is independently hydrogen or an optionally substituted C₁₋₆    aliphatic group;-   each of R⁶ and R^(6′) is independently R, halogen, —CN, —NO₂, —OR,    —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    or —OSi(R)₃; or    -   R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;-   each n is independently 0, 1, 2, 3, or 4;-   each R⁷ is independently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR,    —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    —P(R)₂, —P(OR)₂, —P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂,    —B(OR)₂, or —Si(R)₃; or    -   two R⁷ are taken together with their intervening atoms to form        an optionally substituted 4-7 membered ring having 0-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   each R⁸ is independently —(S)_(m)—R^(x) wherein m is 1-3 and R^(x)    is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or    —S(O)₂N(R)₂; and-   each R⁹ is independently —(S)_(p)—R^(y) wherein p is 1-3 such that    m+p is 2-4 and R^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R,    —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or    -   R⁸ and R⁹ are taken together to form —S—,        —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,        —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,        —(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)—.

In some embodiments, the present invention provides drug-ligandconjugate compounds. In some embodiments, the present invention providesa compound having the structure of formula II:

MLD)s]_(t)   II

or a pharmaceutically acceptable salt thereof, wherein:M is a cell-specific ligand unit;each L is independently a linker unit;each D independently has the structure of formula I-c or I-d,

or a pharmaceutically acceptable salt thereof, wherein:

-   each    is independently a single bond or a double bond, as valency permits;-   each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R,    —S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂;-   each R is independently hydrogen or an optionally substituted group    selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7    membered saturated or partially unsaturated carbocyclic ring, an    8-14 membered bicyclic or polycyclic saturated, partially    unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 3-7 membered saturated or partially unsaturated    heterocyclic ring having 1-3 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic    saturated or partially unsaturated heterocyclic ring having 1-5    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    or an 8-14 membered bicyclic or polycyclic heteroaryl ring having    1-5 heteroatoms independently selected from nitrogen, oxygen, or    sulfur; or:    -   two R groups are optionally taken together with their        intervening atoms to form an optionally substituted 3-14        membered, saturated, partially unsaturated, or aryl ring having,        in addition to the intervening atoms, 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,-   each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,    —[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,    —[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,    —[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or    -   R¹ and R² are taken together with their intervening atoms to        form an optionally substituted 4-7 membered heterocyclic ring        having, in addition to the nitrogen atom to which R¹ is        attached, 0-2 heteroatoms independently selected from oxygen,        nitrogen or sulfur;-   each q is independently 0, 1, 2, 3, or 4;-   each R^(3′) is independently R or an electron-withdrawing group;-   each R⁴ is independently absent when    is a double bond or is independently R or halogen;-   each R⁵ is independently absent when    is a double bond or is independently hydrogen or an optionally    substituted C₁₋₆ aliphatic group;-   each of R⁶ and R^(6′) is independently R, halogen, —CN, —NO₂, —OR,    —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    or —OSi(R)₃; or    -   R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;-   each n is independently 0, 1, 2, 3, or 4;-   each R⁷ is independently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR,    —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    —P(R)₂, —P(OR)₂, —P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂,    —B(OR)₂, or —Si(R)₃; or:    -   two R⁷ are taken together with their intervening atoms to form        an optionally substituted 4-7 membered ring having 0-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   each R⁸ is independently —(S)_(m)—R^(x) wherein m is 1-3 and R^(x)    is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or    —S(O)₂N(R)₂; and-   each R⁹ is independently —(S)_(p)—R^(y) wherein p is 1-3 such that    m+p is 2-4 and R^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R,    —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or    -   R⁸ and R⁹ are taken together to form —S—,        —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,        —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,        —(S)_(m)—S(O)S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)—;-   s is 1-10; and-   t is 1-10.

In some embodiments, the present invention provides a compound havingthe structure of formula II-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, the present invention provides a compound havingthe structure of formula II-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, the present invention provides a compound havingthe structure of formula III:

H-LD)s   III

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, the present invention provides a compound havingthe structure of formula III-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, the present invention provides a compound havingthe structure of formula III-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

2. Definitions

Compounds of the present invention include those described generallyherein, and are further illustrated by the classes, subclasses, andspecies disclosed herein. As used herein, the following definitionsshall apply unless otherwise indicated. For purposes of this invention,the chemical elements are identified in accordance with the PeriodicTable of the Elements, CAS version, Handbook of Chemistry and Physics,93^(rd) Ed. Additionally, general principles of organic chemistry aredescribed in “Organic Chemistry”, 2^(nd) Ed, Thomas N. Sorrell,University Science Books, Sausalito: 2005, and “March's Advanced OrganicChemistry”, 6^(th) Ed., Smith, M. B, and March, J., John Wiley & Sons,New York: 2007, the entire contents of which are hereby incorporated byreference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbon,bicyclic hydrocarbon, or polycyclic hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic (also referred to herein as “carbocycle,”“cycloaliphatic” or “cycloalkyl”), that has, unless otherwise specified,a single point of attachment to the rest of the molecule. Unlessotherwise specified, aliphatic groups contain 1-30 aliphatic carbonatoms. In some embodiments, aliphatic groups contain 1-20 aliphaticcarbon atoms. In other embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitablealiphatic groups include, but are not limited to, linear or branched,substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybridsthereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “cycloaliphatic,” as used herein, refers to saturated orpartially unsaturated cyclic aliphatic monocyclic, bicyclic, orpolycyclic ring systems, as described herein, having from 3 to 14members, wherein the aliphatic ring system is optionally substituted asdefined above and described herein. Cycloaliphatic groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl,cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In someembodiments, the cycloalkyl has 3-6 carbons. The terms “cycloaliphatic,”may also include aliphatic rings that are fused to one or more aromaticor nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl,where the radical or point of attachment is on the aliphatic ring. Insome embodiments, a carbocyclic group is bicyclic. In some embodiments,a carbocyclic group is tricyclic. In some embodiments, a carbocyclicgroup is polycyclic. In some embodiments, “cycloaliphatic” (or“carbocycle” or “cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon,or a C₈-C₁₀ bicyclic hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule, or aC₉-C₁₆ tricyclic hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.

As used herein, the term “alkyl” is given its ordinary meaning in theart and may include saturated aliphatic groups, including straight-chainalkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic)groups, alkyl substituted cycloalkyl groups, and cycloalkyl substitutedalkyl groups. In certain embodiments, a straight chain or branched chainalkyl has about 1-20 carbon atoms in its backbone (e.g., C₁-C₂₀ forstraight chain, C₂-C₂₀ for branched chain), and alternatively, about1-10. In some embodiments, a cycloalkyl ring has from about 3-10 carbonatoms in their ring structure where such rings are monocyclic, bicyclicor polycyclic, and alternatively about 5, 6 or 7 carbons in the ringstructure. In some embodiments, an alkyl group may be a lower alkylgroup, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g.,C₁-C₄ for straight chain lower alkyls).

As used herein, the term “alkenyl” refers to an alkyl group, as definedherein, having one or more double bonds.

As used herein, the term “alkynyl” refers to an alkyl group, as definedherein, having one or more triple bonds.

The term “heteroalkyl” is given its ordinary meaning in the art andrefers to alkyl groups as described herein in which at least one carbonatom, optionally with one or more attached hydrogen atoms, is replacedwith a heteroatom (e.g., oxygen, nitrogen, sulfur, phosphorus, selenium,boron and the like). Examples of heteroalkyl groups include, but are notlimited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino,tetrahydrofuranyl, piperidinyl, morpholinyl, etc. In some embodiments, aheteroatom may be oxidized (e.g., —S(O)—, —S(O)₂—, —N(O)—, —P(O)— andthe like).

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclicor polycyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic and whereineach ring in the system contains 3 to 7 ring members. The term “aryl”may be used interchangeably with the term “aryl ring.” In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,binaphthyl, anthracyl and the like, which may bear one or moresubstituents. Also included within the scope of the term “aryl,” as itis used herein, is a group in which an aromatic ring is fused to one ormore non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms (i.e., monocyclic or bicyclic), in someembodiments 5, 6, 9, or 10 ring atoms. In some embodiments, such ringshave 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. In some embodiments, aheteroaryl is a heterobiaryl group, such as bipyridyl and the like. Theterms “heteroaryl” and “heteroar-”, as used herein, also include groupsin which a heteroaromatic ring is fused to one or more aryl,cycloaliphatic, or heterocyclyl rings, where the radical or point ofattachment is on the heteroaromatic ring. Non-limiting examples includeindolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono- orbicyclic. The term “heteroaryl” may be used interchangeably with theterms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any ofwhich terms include rings that are optionally substituted. The term“heteroaralkyl” refers to an alkyl group substituted by a heteroaryl,wherein the alkyl and heteroaryl portions independently are optionallysubstituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothienyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

The term “heteroatom” means one or more of boron, oxygen, sulfur,selenium, nitrogen, phosphorus, or silicon (including, any oxidized formof nitrogen, sulfur, selenium, phosphorus, or silicon; the quaternizedform of any basic nitrogen; or a substitutable nitrogen of aheterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

The term “halogen” means F, Cl, Br, or I.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogen atoms of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄S(O)R^(∘);—O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘); —(CH₂)₀₋₄CH(OR^(∘))₂;—(CH₂)₀₋₄Ph, which may be substituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Phwhich may be substituted with R^(∘); —CH═CHPh, which may be substitutedwith R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted withR^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR; —SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘); —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂;—P(O)(OR^(∘))R^(∘); —P(O)(OR^(∘))₂; —OP(O)R^(∘) ₂; —OP(O)(OR^(∘))R^(∘);—OP(O)(OR^(∘))₂; —PR^(∘) ₂; —P(OR^(∘))R^(∘); —P(OR^(∘))₂; —OPR^(∘) ₂;—OP(OR^(∘))R^(∘); —OP(OR^(∘))₂; —SiR^(∘) ₃; —OSiR^(∘) ₃; —SeR^(∘);—(CH₂)₀₋₄SeSeR^(∘); —B(R^(∘))₂, —B(OR^(∘))₂, —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂; wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a suitable carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “chiral” is given its ordinary meaning in the art and refers toa molecule that is not superimposable with its mirror image, wherein theresulting non-superimposable mirror images are known as “enantiomers”and are labeled as either an (R) enantiomer or an (S) enantiomer.Typically, chiral molecules lack a plane of symmetry.

The term “achiral” is given its ordinary meaning in the art and refersto a molecule that is superimposable with its mirror image. Typically,achiral molecules possess a plane of symmetry.

As used herein, the term “electron-withdrawing group” is given itsordinary meaning in the art and refers to an atom or group that drawselectron density from a neighboring atom or group, usually by resonanceand/or inductive effects. In some embodiments, an electron-withdrawinggroup withdraws electron density from an aromatic ring system byresonance and/or inductive effects. In some embodiments, anelectron-withdrawing group withdraws electron density from an aromaticring system by resonance and inductive effects. In some embodiments, anelectron-withdrawing group lowers the electron density of an aromaticring system such as phenyl. Exemplary electron-withdrawing groups areextensively described in the art, including but not limited to halogen,carbonyl moieties (e.g., aldehyde and ketone groups), —COOH and itsderivatives (e.g., ester and amide moieties), protonated amines,quaternary ammonium groups, —CN, —NO₂, —S(O)— moieties, —P(O)— moietiesand —S(O)₂— moieties. In some embodiments, an electron-withdrawing groupcomprises one or more —C(O)—, —C(═N—)—, —C(S)—, —S(O)—, —S(O)₂— or—P(O)— groups, and is connected to the rest of a molecule via one ormore —C(O)—, —C(═N—)—, —C(S)—, —S(O)—, —S(O)₂— or —P(O)— groups. In someembodiments, an electron-withdrawing group is halogen. In someembodiments, an electron-withdrawing group is —F. In some embodiments,an electron-withdrawing group is —Cl. In some embodiments, anelectron-withdrawing group is —Br. In some embodiments, anelectron-withdrawing group is —I. In some embodiments, hydrogen is usedas reference and regarded as having no effect.

The phrase “protecting group,” as used herein, refers to temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. A “Siprotecting group” is a protecting group comprising a Si atom, such asSi-trialkyl (e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl),Si-triaryl, Si-alkyl-diphenyl (e.g., t-butyldiphenylsilyl), orSi-aryl-dialkyl (e.g., Si-phenyldialkyl). Generally, a Si protectinggroup is attached to an oxygen atom. The field of protecting groupchemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. ProtectiveGroups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Suchprotecting groups (and associated protected moieties) are described indetail below.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, propionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl ethers includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Alkoxyalkyl ethers include acetals such asmethoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethersinclude benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl,O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (—NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino (—NHAlloc),benzyloxycarbonylamino (—NHCBZ), allylamino, benzylamino (—NHBn),fluorenylmethylcarbonyl (—NHFmoc), formamido, acetamido,chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₆ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention.

Unless otherwise stated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹¹C- or ¹³C- or¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays.

As used herein and in the claims, the singular forms “a”, “an”, and“the” include the plural reference unless the context clearly indicatesotherwise. Thus, for example, a reference to “a compound” includes aplurality of such compounds.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within an organism (e.g., animal, plant,and/or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, and/or microbe).

The phrases “parenteral administration” and “administered parenterally”as used herein have their art-understood meaning referring to modes ofadministration other than enteral and topical administration, usually byinjection, and include, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

As used herein, the term “pharmaceutical composition” refers to anactive agent, formulated together with one or more pharmaceuticallyacceptable carriers. In some embodiments, active agent is present inunit dose amount appropriate for administration in a therapeutic regimenthat shows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. In some embodiments, pharmaceutical compositions may bespecially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar, buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically acceptable salt”, as used herein, refers tosalts of such compounds that are appropriate for use in pharmaceuticalcontexts, i.e., salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In someembodiments, pharmaceutically acceptable salt include, but are notlimited to, nontoxic acid addition salts, which are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. In some embodiments, pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like. Insome embodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

A general, a “prodrug,” as that term is used herein and as is understoodin the art, is an entity that, when administered to an organism, ismetabolized in the body to deliver an active (e.g., therapeutic ordiagnostic) agent of interest. Typically, such metabolism involvesremoval of at least one “prodrug moiety” so that the active agent isformed. Various forms of “prodrugs” are known in the art. For examplesof such prodrug moieties, see:

-   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and    Methods in Enzymology, 42:309-396, edited by K. Widder, et al.    (Academic Press, 1985);-   b) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,    2011);-   c) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,    2011);-   d) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen;-   e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H.    Bundgaard, p. 113-191 (1991);-   f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);-   g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285    (1988); and-   h) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

As with other compounds described herein, prodrugs may be provided inany of a variety of forms, e.g., crystal forms, salt forms etc. In someembodiments, prodrugs are provided as pharmaceutically acceptable saltsthereof.

As used herein, the term “protein” refers to a polypeptide (i.e., astring of at least two amino acids linked to one another by peptidebonds). In some embodiments, proteins include only naturally-occurringamino acids. In some embodiments, proteins include one or morenon-naturally-occurring amino acids (e.g., moieties that form one ormore peptide bonds with adjacent amino acids). In some embodiments, oneor more residues in a protein chain contain a non-amino-acid moiety(e.g., a glycan, etc). In some embodiments, a protein includes more thanone polypeptide chain, for example linked by one or more disulfide bondsor associated by other means. In some embodiments, proteins contain1-amino acids, d-amino acids, or both; in some embodiments, proteinscontain one or more amino acid modifications or analogs known in theart. Useful modifications include, e.g., terminal acetylation,amidation, methylation, etc. The term “peptide” is generally used torefer to a polypeptide having a length of less than about 100 aminoacids, less than about 50 amino acids, less than 20 amino acids, or lessthan 10 amino acids. In some embodiments, proteins are antibodies,antibody fragments, biologically active portions thereof, and/orcharacteristic portions thereof.

As used herein, the term “subject” or “test subject” refers to anyorganism to which a provided compound or composition is administered inaccordance with the present invention e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.). In some embodiments, asubject may be suffering from, and/or susceptible to a disease,disorder, and/or condition. In some embodiments, a subject is human.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and/or chemical phenomena.

An individual who is “suffering from” a disease, disorder, and/orcondition has been diagnosed with and/or displays one or more symptomsof a disease, disorder, and/or condition

An individual who is “susceptible to” a disease, disorder, and/orcondition is one who has a higher risk of developing the disease,disorder, and/or condition than does a member of the general public. Insome embodiments, an individual who is susceptible to a disease,disorder and/or condition may not have been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

The phrases “systemic administration,” “administered systemically,”“peripheral administration,” and “administered peripherally” as usedherein have their art-understood meaning referring to administration ofa compound or composition such that it enters the recipient's system.

As used herein, the phrase “therapeutic agent” refers to any agent that,when administered to a subject, has a therapeutic effect and/or elicitsa desired biological and/or pharmacological effect. In some embodiments,a therapeutic agent is any substance that can be used to alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition.

As used herein, the term “therapeutically effective amount” means anamount of a substance (e.g., a therapeutic agent, composition, and/orformulation) that elicits a desired biological response whenadministered as part of a therapeutic regimen. In some embodiments, atherapeutically effective amount of a substance is an amount that issufficient, when administered to a subject suffering from or susceptibleto a disease, disorder, and/or condition, to treat, diagnose, prevent,and/or delay the onset of the disease, disorder, and/or condition. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a substance may vary depending on such factors asthe desired biological endpoint, the substance to be delivered, thetarget cell or tissue, etc. For example, the effective amount ofcompound in a formulation to treat a disease, disorder, and/or conditionis the amount that alleviates, ameliorates, relieves, inhibits,prevents, delays onset of, reduces severity of and/or reduces incidenceof one or more symptoms or features of the disease, disorder, and/orcondition. In some embodiments, a therapeutically effective amount isadministered in a single dose; in some embodiments, multiple unit dosesare required to deliver a therapeutically effective amount.

As used herein, the term “treat,” “treatment,” or “treating” refers toany method used to partially or completely alleviate, ameliorate,relieve, inhibit, prevent, delay onset of, reduce severity of, and/orreduce incidence of one or more symptoms or features of a disease,disorder, and/or condition. Treatment may be administered to a subjectwho does not exhibit signs of a disease, disorder, and/or condition. Insome embodiments, treatment may be administered to a subject whoexhibits only early signs of the disease, disorder, and/or condition,for example for the purpose of decreasing the risk of developingpathology associated with the disease, disorder, and/or condition.

The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

As used herein, the term “wild-type” has its art-understood meaning thatrefers to an entity having a structure and/or activity as found innature in a “normal” (as contrasted with mutant, diseased, altered, etc)state or context. Those of ordinary skill in the art will appreciatethat wild type genes and polypeptides often exist in multiple differentforms (e.g., alleles).

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Therelevant intended purpose may be objective (i.e., measurable by sometest or marker) or subjective (i.e., subject gives an indication of orfeels an effect). In some embodiments, a therapeutically effectiveamount is an amount that, when administered to a population of subjectsthat meet certain clinical criteria for a disease or disorder (forexample, as determined by symptoms manifested, diseaseprogression/stage, genetic profile, etc.), a statistically significanttherapeutic response is obtained among the population. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular pharmaceutical agent, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents. Insome embodiments, the specific therapeutically effective amount (and/orunit dose) for any particular patient may depend upon a variety offactors including the disorder being treated and the severity of thedisorder; the activity of the specific pharmaceutical agent employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and/or rate of excretion or metabolism of the specificpharmaceutical agent employed; the duration of the treatment; and likefactors as is well known in the medical arts. Those of ordinary skill inthe art will appreciate that in some embodiments of the invention, aunit dosage may be considered to contain an effective amount if itcontains an amount appropriate for administration in the context of adosage regimen correlated with a positive outcome.

3. Description of Certain Embodiments of the Invention

In some embodiments, the present invention provides a compound havingthe structure of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides a compound havingthe structure of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides drug-ligandconjugate compounds. In some embodiments, the present invention providesa compound having the structure of formula II:

MLD)s]_(t)   II

or a pharmaceutically acceptable salt thereof, wherein:M is a cell-specific ligand unit;each L is independently a linker unit;each D independently has the structure of formula I-c or I-d,

or a pharmaceutically acceptable salt thereof, wherein:

-   each    is independently a single bond or a double bond, as valency permits;-   each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R,    —S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂;-   each R is independently hydrogen or an optionally substituted group    selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7    membered saturated or partially unsaturated carbocyclic ring, an    8-14 membered bicyclic or polycyclic saturated, partially    unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, a 3-7 membered saturated or partially unsaturated    heterocyclic ring having 1-3 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic    saturated or partially unsaturated heterocyclic ring having 1-5    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    or an 8-14 membered bicyclic or polycyclic heteroaryl ring having    1-5 heteroatoms independently selected from nitrogen, oxygen, or    sulfur; or:    -   two R groups are optionally taken together with their        intervening atoms to form an optionally substituted 3-14        membered, saturated, partially unsaturated, or aryl ring having,        in addition to the intervening atoms, 0-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,-   each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,    —[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,    —[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,    —[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or    -   R¹ and R² are taken together with their intervening atoms to        form an optionally substituted 4-7 membered heterocyclic ring        having, in addition to the nitrogen atom to which R¹ is        attached, 0-2 heteroatoms independently selected from oxygen,        nitrogen or sulfur;-   each q is independently 0, 1, 2, 3, or 4;-   each R^(3′) is independently R or an electron-withdrawing group;-   each R⁴ is independently absent when    is a double bond or is independently R or halogen;-   each R⁵ is independently absent when    is a double bond or is independently hydrogen or an optionally    substituted C₁₋₆ aliphatic group;-   each of R⁶ and R^(6′) is independently R, halogen, —CN, —NO₂. —OR,    —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    or —OSi(R)₃; or    -   R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;-   each n is independently 0, 1, 2, 3, or 4;-   each R⁷ is independently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR,    —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,    —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R,    —P(R)₂, —P(OR)₂, —P(OX)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂,    —B(OR)₂, or —Si(R)₃; or    -   two R⁷ are taken together with their intervening atoms to form        an optionally substituted 4-7 membered ring having 0-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   each R⁸ is independently —(S)_(m)—R^(x) wherein m is 1-3 and R^(x)    is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or    —S(O)₂N(R)₂; and-   each R⁹ is independently —(S)_(p)—R^(y) wherein p is 1-3 such that    m+p is 2-4 and R^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R,    —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or    -   R⁸ and R⁹ are taken together to form —S—,        —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,        —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,        —(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)—;-   s is 1-10; and-   t is 1-10.

In some embodiments, the present invention provides a compound havingthe structure of formula II-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides a compound havingthe structure of formula II-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides a compound havingthe structure of formula III:

H-LD)s   III

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides a compound havingthe structure of formula III-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, the present invention provides a compound havingthe structure of formula III-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

As generally defined above,

is a single bond or a double bond, as valency permits. In someembodiments,

is a single bond. In some embodiments,

is a double bond. In some embodiments, there are two or more

in a provided compound, and at least one

is a single bond, and at least one

is a double bond. In some other embodiments, there are two or more

in a provided compound, and each

is a single bond. In some other embodiments, there are two or more

in a provided compound, and each

is a double bond.

In some embodiments,

is a single bond, R⁴ is R or halogen, and R⁵ is hydrogen or anoptionally substituted C₁₋₆ aliphatic. In some embodiments,

is a double bond, R⁴ is absent and R⁵ is absent.

As generally defined above, each R¹ is independently R, —C(O)R,—C(O)N(R)₂, —S(O)R, —S(O)₂R, —S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂, or R¹and R² are taken together with their intervening atoms to form anoptionally substituted 4-7 membered heterocyclic ring having, inaddition to the nitrogen atom to which R¹ is attached, 0-2 heteroatomsindependently selected from oxygen, nitrogen or sulfur. In someembodiments, each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R,—S(O)₂R, —S(O)₂OR, —CH₂OR, or —S(O)₂N(R)₂, or R¹ and R² are takentogether with their intervening atoms to form an optionally substituted4-7 membered heterocyclic ring having, in addition to the nitrogen atomto which R¹ is attached, 0-2 heteroatoms independently selected fromoxygen, nitrogen or sulfur. In some embodiments, each R¹ isindependently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R, —S(O)₂OR,—C(R)₂OR, or —S(O)₂N(R)₂, or R¹ and R² are taken together with theirintervening atoms to form an optionally substituted 4-7 memberedheterocyclic ring having, in addition to the nitrogen atom to which R¹is attached, 0-2 heteroatoms independently selected from oxygen,nitrogen or sulfur. In some embodiments, R¹ is R, —C(O)R, —C(O)N(R)₂,—S(O)R, —S(O)₂R, —S(O)₂OR, or —S(O)₂N(R)₂. In some embodiments, R¹ is R.In some embodiments, R¹ is —C(O)R. In some embodiments, R¹ is—C(O)N(R)₂. In some embodiments, R¹ is —S(O)R. In some embodiments, R¹is —S(O)₂R. In some embodiments, R¹ is —S(O)₂OR. In some embodiments, R¹is —C(R)₂OR. In some embodiments, R¹ is —CH₂OR. In some embodiments, R¹is —S(O)₂N(R)₂. In some embodiments, a provided compound has more thanone R¹ groups. In some embodiments, each R¹ of a provided compound isthe same. In some embodiments, at least one R¹ is different from theother R¹.

In some embodiments, R¹ is R. In some embodiments, R¹ is hydrogen. Insome embodiments, R¹ is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R¹ is optionally substituted C₁₋₁₀ aliphatic. In someembodiments, R¹ is optionally substituted C₁₋₆ aliphatic. In someembodiments, R¹ is optionally substituted C₁₋₆ alkyl. In someembodiments, R¹ is optionally substituted hexyl, pentyl, butyl, propyl,ethyl or methyl. In some embodiments, R¹ is optionally substitutedhexyl. In some embodiments, R¹ is optionally substituted pentyl. In someembodiments, R¹ is optionally substituted butyl. In some embodiments, R¹is optionally substituted propyl. In some embodiments, R¹ is optionallysubstituted ethyl. In some embodiments, R¹ is optionally substitutedmethyl. In some embodiments, R¹ is hexyl. In some embodiments, R¹ ispentyl. In some embodiments, R¹ is butyl. In some embodiments, R¹ ispropyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ ismethyl. In some embodiments, R¹ is isopropyl. In some embodiments, R¹ isn-propyl. In some embodiments, R¹ is tert-butyl. In some embodiments, R¹is sec-butyl. In some embodiments, R¹ is n-butyl. In some embodiments,R¹ is benzyloxymethyl. In some embodiments, R¹ is benzyl. In someembodiments, R¹ is allyl.

In some embodiments, R¹ is methyl, R³ is other than hydrogen, Boc(tert-butyloxycarbonyl) and CF₃C(O)—. In some embodiments, R¹ is methyl,R³ is other than hydrogen and CF₃C(O)—. In some embodiments, R¹ ismethyl, R³ is other than hydrogen. In some embodiments, R¹ is methyl, R³is other than hydrogen, Boc (tert-butyloxycarbonyl) and CF₃C(O)—. Insome embodiments, R¹ is other than methyl.

Exemplary R¹ groups are depicted below.

In some embodiments, in a provided compound of formula II, D isconnected to L through R¹. In some embodiments, R¹ comprises an —OH,—NHR or —SH group for conjugation. In some embodiments, R¹ comprises an—OH group, and D is connected to L through the —OH group. In someembodiments, the —OH group reacts with a functional group in L or M toform, for example, an ether, ester, carbamate or carbonate ester. Insome embodiments, R¹ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R¹ comprises a —NH₂ group.In some embodiments, R¹ comprises a —NHR group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R¹comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

As generally defined above, each R is independently hydrogen or anoptionally substituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated,partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, ortwo R⁷ are taken together with their intervening atoms to form anoptionally substituted 4-7 membered ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, each R is independently hydrogen or an optionallysubstituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl,phenyl, a 3-7 membered saturated or partially unsaturated carbocyclicring, an 8-10 membered bicyclic saturated, partially unsaturated or arylring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, a 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, a7-10 membered bicyclic saturated or partially unsaturated heterocyclicring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or two R groups are optionally taken together with their interveningatoms to form an optionally substituted 3-14 membered, saturated,partially unsaturated, or aryl ring having, in addition to theintervening atoms, 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, each R is independently hydrogen or an optionallysubstituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl,phenyl, a 3-7 membered saturated or partially unsaturated carbocyclicring, an 8-14 membered bicyclic or polycyclic saturated, partiallyunsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, a 3-7 membered saturated or partially unsaturated heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic saturated orpartially unsaturated heterocyclic ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or an 8-14membered bicyclic or polycyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is hydrogen or an optionally substituted group selectedfrom C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 memberedsaturated or partially unsaturated carbocyclic ring, an 8-10 memberedbicyclic saturated, partially unsaturated or aryl ring, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, a 7-10 membered bicyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, two R groups are optionally taken together with theirintervening atoms to form an optionally substituted 3-14 membered,saturated, partially unsaturated, or aryl ring having, in addition tothe intervening atoms, 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is optionally substituted C₁₋₂₀ aliphatic. Insome embodiments, R is optionally substituted C₁₋₁₅ aliphatic. In someembodiments, R is optionally substituted C₁₋₁₀ aliphatic. In someembodiments, R is optionally substituted C₁₋₆ aliphatic. In someembodiments, R is optionally substituted C₁₋₆ alkyl. In someembodiments, R is optionally substituted hexyl, pentyl, butyl, propyl,ethyl or methyl. In some embodiments, R is optionally substituted hexyl.In some embodiments, R is optionally substituted pentyl. In someembodiments, R is optionally substituted butyl. In some embodiments, Ris optionally substituted propyl. In some embodiments, R is optionallysubstituted ethyl. In some embodiments, R is optionally substitutedmethyl. In some embodiments, R is hexyl. In some embodiments, R ispentyl. In some embodiments, R is butyl. In some embodiments, R ispropyl. In some embodiments, R is ethyl. In some embodiments, R ismethyl. In some embodiments, R is isopropyl. In some embodiments, R isn-propyl. In some embodiments, R is tert-butyl. In some embodiments, Ris sec-butyl. In some embodiments, R is n-butyl. In some embodiments, Ris benzyloxymethyl. In some embodiments, R is benzyl. In someembodiments, R is allyl. In some embodiments, R is not hydrogen. In someembodiments, R is not alkyl.

In some embodiments, R is optionally substituted C₁₋₂₀ heteroalkyl. Insome embodiments, R is optionally substituted C₁₋₂₀ heteroalkyl having1-6 heteroatoms independently selected from nitrogen, sulfur, phosphorusselenium, silicon or boron. In some embodiments, R is optionallysubstituted C₁₋₂₀ heteroalkyl having 1-6 heteroatoms independentlyselected from nitrogen, sulfur, phosphorus, selenium, silicon or boron,optionally including one or more oxidized forms of nitrogen, sulfur,phosphorus, selenium, silicon or boron. In some embodiments, R isoptionally substituted C₁₋₂₀ heteroalkyl comprising 1-6 groupsindependently selected from

—N═, ≡N, —S—, —S(O)—, —S(O)₂—, —O—, ═O,

—Se—, —Se(O)—, and

In some embodiments, R is not heteroalkyl. In some embodiments, R ismethoxymethyl. In some embodiments, R is benzyloxymethyl.

In some embodiments, R is optionally substituted phenyl. In someembodiments, R is optionally substituted phenyl wherein one or moresubstituents are halogen. In some embodiments, R is optionallysubstituted phenyl wherein one or more substituents are —F. In someembodiments, R is optionally substituted phenyl wherein one or moresubstituents are —Cl. In some embodiments, R is optionally substitutedphenyl wherein one or more substituents are —Br. In some embodiments, Ris optionally substituted phenyl wherein one or more substituents are—I. In some embodiments, R is phenyl.

In some embodiments, R is an optionally substituted 3-7 memberedsaturated or partially unsaturated carbocyclic ring. In someembodiments, R is an optionally substituted 3-membered saturated orpartially unsaturated carbocyclic ring. In some embodiments, R is anoptionally substituted 4-membered saturated or partially unsaturatedcarbocyclic ring. In some embodiments, R is an optionally substituted5-membered saturated or partially unsaturated carbocyclic ring. In someembodiments, R is an optionally substituted 6-membered saturated orpartially unsaturated carbocyclic ring. In some embodiments, R is anoptionally substituted 7-membered saturated or partially unsaturatedcarbocyclic ring. In some embodiments, R is optionally substitutedcycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments,R is optionally substituted cyclohexyl. In some embodiments, R iscyclohexyl. In some embodiments, R is optionally substitutedcyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments,R is optionally substituted cyclobutyl. In some embodiments, R iscyclobutyl. In some embodiments, R is optionally substitutedcyclopropyl. In some embodiments, R is cyclopropyl.

In some embodiments, R is an optionally substituted 8-14 memberedbicyclic or polycyclic saturated, partially unsaturated or aryl ring. Insome embodiments, R is an optionally substituted 8-14 membered bicyclicor polycyclic saturated ring. In some embodiments, R is an optionallysubstituted 8-14 membered bicyclic or polycyclic partially saturatedring. In some embodiments, R is an optionally substituted 8-14 memberedbicyclic or polycyclic aryl ring. In some embodiments, R is anoptionally substituted 8-10 membered bicyclic saturated, partiallyunsaturated or aryl ring. In some embodiments, R is an optionallysubstituted 8-10 membered bicyclic saturated ring. In some embodiments,R is an optionally substituted 8-10 membered bicyclic partiallyunsaturated ring. In some embodiments, R is an optionally substituted8-10 membered bicyclic aryl ring. In some embodiments, R is optionallysubstituted naphthyl. In some embodiments, R is optionally substitutedanthracenyl. In some embodiments, R is optionally substituted9-anthracenyl.

In some embodiments, R is optionally substituted biaryl wherein eacharyl group is independently an optionally substituted group selectedfrom phenyl, 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10 membered bicyclic aryl ring, or an 8-10 membered bicyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is optionallysubstituted biaryl wherein each aryl group is independently anoptionally substituted group selected from phenyl, 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic aryl ring,or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and wherein atleast one aryl group is optionally substituted phenyl. In someembodiments, R is optionally substituted biaryl wherein each aryl groupis independently an optionally substituted group selected from phenyl,5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered bicyclic aryl ring, or an 8-10 membered bicyclic heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur, and wherein at least one aryl group is an optionallysubstituted 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is optionally substituted biaryl wherein each arylgroup is independently an optionally substituted group selected fromphenyl, 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered bicyclic aryl ring, or an 8-10 membered bicyclic heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur, and wherein at least one aryl group is an optionallysubstituted 8-10 membered bicyclic aryl ring. In some embodiments, R isoptionally substituted biaryl wherein each aryl group is independentlyan optionally substituted group selected from phenyl, 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic aryl ring,or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and wherein atleast one aryl group is optionally substituted naphthyl. In someembodiments, R is optionally substituted biaryl wherein each aryl groupis independently an optionally substituted group selected from phenyl,5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered bicyclic aryl ring, or an 8-10 membered bicyclic heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur, and wherein at least one aryl group is an optionallysubstituted 8-10 membered bicyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is optionally substituted biaryl wherein each arylgroup is independently optionally substituted phenyl. In someembodiments, R is optionally substituted biaryl wherein each aryl groupis independently optionally substituted phenyl, or an optionallysubstituted 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen. In someembodiments, R is optionally substituted biaryl wherein each aryl groupis independently an optionally substituted 8-10 membered bicyclic arylring. In some embodiments, R is optionally substituted biaryl whereinone aryl group is optionally substituted naphthyl, and the other arylgroup is independently an optionally substituted 8-10 membered bicyclicaryl ring. In some embodiments, R is optionally substituted biarylwherein each aryl group is optionally substituted naphthyl. In someembodiments, R is optionally substituted biaryl wherein one aryl groupis optionally substituted naphthyl, and the other aryl group is anoptionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is asubstituted 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is an unsubstituted 5-6 membered monocyclicheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having one heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R is selected from optionallysubstituted pyrrolyl, furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having two heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having one nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Exemplary R groupsinclude but are not limited to optionally substituted pyrazolyl,imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having three heteroatoms independently selected fromnitrogen, oxygen, or sulfur. Exemplary R groups include but are notlimited to optionally substituted triazolyl, oxadiazolyl orthiadiazolyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having four heteroatoms independently selected fromnitrogen, oxygen, or sulfur. Exemplary R groups include but are notlimited to optionally substituted tetrazolyl, oxatriazolyl andthiatriazolyl.

In some embodiments, R is an optionally substituted 6-memberedheteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is anoptionally substituted 6-membered heteroaryl ring having 1-3 nitrogenatoms. In other embodiments, R is an optionally substituted 6-memberedheteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is anoptionally substituted 6-membered heteroaryl ring having four nitrogenatoms. In some embodiments, R is an optionally substituted 6-memberedheteroaryl ring having three nitrogen atoms. In some embodiments, R isan optionally substituted 6-membered heteroaryl ring having two nitrogenatoms. In certain embodiments, R is an optionally substituted 6-memberedheteroaryl ring having one nitrogen atom. Exemplary R groups include butare not limited to optionally substituted pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

In some embodiments, R is an optionally substituted 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is optionally substituted 3-membered heterocyclicring having one heteroatom selected from nitrogen, oxygen or sulfur.Exemplary R groups include but are not limited to optionally substitutedaziridinyl, thiiranyl or oxiranyl. In some embodiments, R is optionallysubstituted 4-membered heterocyclic ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary Rgroups include but are not limited to optionally substituted azetidinyl,oxetanyl, thietanyl, oxazetidinyl, thiazetidinyl, or diazetidinyl. Insome embodiments, R is optionally substituted 5-membered heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Exemplary R groups include but are not limited tooptionally substituted pyrrolidinyl, tetrahydrofuranyl,tetrahydrothienyl, oxazolidinyl, dioxolanyl, oxathiolanyl,thiazolidinyl, dithiolanyl, imidazolidinyl, isothiazolidinyl,pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, triazolidinyl,oxadiazolidinyl, thiadiazolidinyl, oxadiazolidinyl, dioxazolidinyl,oxathiazolidinyl, thiadiazolidinyl or dithiazolidinyl. In someembodiments, R is optionally substituted 6-membered heterocyclic ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. Exemplary R groups include but are not limited to optionallysubstituted piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, dioxanyl,oxathianyl, triazinanyl, oxadiazinanyl, thiadiazinanyl, dithiazinanyl,dioxazinanyl, oxathiazinanyl, oxadithianyl, trioxanyl, dioxathianyl ortrithianyl. In some embodiments, R is optionally substituted 7-memberedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. Exemplary R groups include but are notlimited to optionally substituted azepanyl, oxepanyl, thiepanyl,diazepanyl, oxazepanyl, thiazepanyl, dioxepanyl, oxathiepanyl,dithiepanyl, triazepanyl, oxadiazepanyl, thiadiazepanyl, dioxazepanyl,oxathiazepanyl, dithiazepanyl, trioxepanyl, dioxathiepanyl,oxadithiepanyl or trithiepanyl.

In certain embodiments, R is an optionally substituted 5-7 memberedpartially unsaturated monocyclic ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R is an optionally substituted 5-6 membered partiallyunsaturated monocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R isan optionally substituted 5-membered partially unsaturated monocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Exemplary R groups include but are not limited tooptionally substituted dihydroimidazolyl, dihydrothiazolyl,dihydrooxazolyl, or oxazolinyl. In certain embodiments, R is anoptionally substituted 6-membered partially unsaturated monocyclic ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. Exemplary R groups include but are not limited to optionallysubstituted dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl,tetrahydropyrimidinyl, dihydropyrazinyl, tetrohydropyrazinyl,dihydrotriazinyl, tetrahydrotriazinyl, dihydrodioxinyl,dihydrooxathiinyl, dihydrooxazinyl, dihydrodithiine, dihydrothiazine,dioxinyl, oxathiinyl, oxazinyl, dithiinyl, or thiazinyl. In certainembodiments, R is an optionally substituted 7-membered partiallyunsaturated monocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Exemplary R groups includebut are not limited to optionally substituted azepiyl, oxepinyl,thiepinyl, diazepinyl, oxazepinyl, thiazepinyl, triazepinyl,oxadiazepinyl, thiadiazepinyl, dihydroazepiyl, dihydrooxepinyl,dihydrothiepinyl, dihydrodiazepinyl, dihydrooxazepinyl,dihydrothiazepinyl, dihydrotriazepinyl, dihydrooxadiazepinyl,dihydrothiadiazepinyl, tetrahydroazepiyl, tetrahydrooxepinyl,tetrahydrothiepinyl, tetrahydrodiazepinyl, tetrahydrooxazepinyl,tetrahydrothiazepinyl, tetrahydrotriazepinyl, tetrahydrooxadiazepinyl,or tetrahydrothiadiazepinyl.

In certain embodiments, R is optionally substituted oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl, aziridineyl,azetidineyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl,tetrahydrothienyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl,oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl,dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl,dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl,diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl,pyrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl,tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl,oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl,oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl,thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl,imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl,dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothienyl, or tetrahydrothiopyranyl.

In some embodiments, R is an optionally substituted 7-14 memberedbicyclic or polycyclic saturated or partially unsaturated heterocyclicring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R is an optionally substituted7-10 membered bicyclic saturated or partially unsaturated heterocyclicring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R is optionally substitutedindolinyl. In some embodiments, R is optionally substitutedisoindolinyl. In some embodiments, R is optionally substituted 1, 2, 3,4-tetrahydroquinolinyl. In some embodiments, R is optionally substituted1, 2, 3, 4-tetrahydroisoquinolinyl. In some embodiments, R is anoptionally substituted azabicyclo[3.2.1]octanyl.

In some embodiments, R is an optionally substituted 8-14 memberedbicyclic or polycyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 8-14 membered bicyclic ortricyclic heteroaryl ring having 1-5 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is an 8-10membered bicyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R is an optionally substituted5,6-fused heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving two heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is optionally substituted1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl,4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl,thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl,pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments,R is an optionally substituted 5,6-fused heteroaryl ring having threeheteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is optionally substituted dihydropyrroloimidazolyl,1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl,4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl,thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl,1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl. Insome embodiments, R is an optionally substituted 5,6-fused heteroarylring having four heteroatoms independently selected from nitrogen,oxygen, or sulfur. In some embodiments, R is an optionally substituted5,6-fused heteroaryl ring having five heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In other embodiments, R is an optionally substituted5,6-fused heteroaryl ring having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having one heteroatomindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is optionally substituted indolyl. In some embodiments, Ris optionally substituted benzofuranyl. In some embodiments, R isoptionally substituted benzo[b]thienyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having two heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is optionally substituted azaindolyl. In someembodiments, R is optionally substituted benzimidazolyl. In someembodiments, R is optionally substituted benzothiazolyl. In someembodiments, R is optionally substituted benzoxazolyl. In someembodiments, R is an optionally substituted indazolyl. In certainembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving three heteroatoms independently selected from nitrogen, oxygen,or sulfur. In some embodiments, R is optionally substitutedoxazolopyridinyl, thiazolopyridinyl or imidazopyridinyl. In certainembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving four heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is optionally substituted purinyl,oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl,thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl,thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R isan optionally substituted 5,6-fused heteroaryl ring having fiveheteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving one heteroatom selected from nitrogen, oxygen, or sulfur. In someembodiments, R is optionally substituted quinolinyl. In someembodiments, R is optionally substituted isoquinolinyl. In someembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving two heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is optionally substituted quinazolinyl,phthalazinyl, quinoxalinyl or naphthyridinyl. In some embodiments, R isan optionally substituted 6,6-fused heteroaryl ring having threeheteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is optionally substituted pyridopyrimidinyl,pyridopyrdazinyl, pyridopyrazinyl, or benzotriazinyl. In someembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving four heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is optionally substitutedpyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl,pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl. Insome embodiments, R is an optionally substituted 6,6-fused heteroarylring having five heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, R is optionally substituted heterobiaryl whereineach heteroaryl group is independently an optionally substituted groupselected from a 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is optionally substituted heterobiaryl wherein each arylgroup is an optionally substituted 8-10 membered bicyclic heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, two R groups are optionally taken together withtheir intervening atoms to form an optionally substituted 3-14 membered,saturated, partially unsaturated, or aryl ring having, in addition tothe intervening atoms, 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, two R groups on thesame atom are optionally taken together with the atom to which they areattached to form an optionally substituted 3-14 membered, saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, two Rgroups on the same carbon atom are optionally taken together with thecarbon atom to form an optionally substituted 3-14 membered, saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, two Rgroups on the same nitrogen atom are optionally taken together with thenitrogen atom to form an optionally substituted 3-14 membered,saturated, partially unsaturated, or aryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, two R groups on the same sulfur atom are optionally takentogether with the sulfur atom to form an optionally substituted 3-14membered, saturated, partially unsaturated, or aryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, two R groups on the same oxygen atom are optionallytaken together with the oxygen atom to form an optionally substituted3-14 membered, saturated, partially unsaturated, or aryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, two R groups on the same phosphorus atom areoptionally taken together with the phosphorus atom to form an optionallysubstituted 3-14 membered, monocyclic or bicyclic, saturated, partiallyunsaturated, or aryl ring having, in addition to the phosphorus atom,0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, two R groups are optionally taken together withtheir intervening atoms to form an optionally substituted 3-14 membered,saturated, partially unsaturated, or aryl ring having, in addition tothe intervening atoms, 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein the two R groups are attached totwo different atoms.

In some embodiments, two R groups are optionally taken together withtheir intervening atoms to form an optionally substituted 3-14 membered,saturated, partially unsaturated, or aryl ring having, in addition tothe intervening atoms, 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, two R groups are takentogether to form an optionally substituted saturated ring. In someembodiments, two R groups are taken together to form an optionallysubstituted partially unsaturated ring. In some embodiments, two Rgroups are taken together to form an optionally substituted carbocyclicring. In some embodiments, two R groups are taken together to form anoptionally substituted aryl ring. In some embodiments, two R groups aretaken together to form an optionally substituted phenyl ring. In someembodiments, two R groups are taken together to form an optionallysubstituted heterocyclic ring. In some embodiments, two R groups aretaken together to form an optionally substituted heteroaryl ring.

In some embodiments, a ring formed by taking two R groups together ismonocyclic, bicyclic or tricyclic. In some embodiments, a ring formed bytaking two R groups together is monocyclic. In some embodiments, a ringformed by taking two R groups together is bicyclic. In some embodiments,a ring formed by taking two R groups together is monocyclic or bicyclic.In some embodiments, a ring formed by taking two R groups together istricyclic. In some embodiments, a ring formed by taking two R groupstogether is monocyclic, bicyclic or tricyclic.

As generally defined above, each R² is independently R, —[C(R)₂]_(q)—OR,—[C(R)₂]_(q)—N(R)₂, —[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃,—[C(R)₂]_(q)—OC(O)R, —[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,—[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂, or R¹ and R² aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered heterocyclic ring having, in addition to thenitrogen atom to which R¹ is attached, 0-2 heteroatoms independentlyselected from oxygen, nitrogen or sulfur. In some embodiments, R² is R,—[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂, —[C(R)₂]_(q)—SR,—[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R, —[C(R)₂]_(q)—OC(O)OR,—[C(R)₂]_(q)—OC(O)N(R)₂, —[C(R)₂]_(q)—OC(O)N(R)—SO₂R or—[C(R)₂]_(q)—OP(OR)₂. In some embodiments, R¹ and R² are taken togetherwith their intervening atoms to form an optionally substituted 4-7membered heterocyclic ring having, in addition to the nitrogen atom towhich R¹ is attached, 0-2 heteroatoms independently selected fromoxygen, nitrogen or sulfur.

In some embodiments, R² is R. In some embodiments, R² is hydrogen. Insome embodiments, R² is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R² is optionally substituted C₁₋₁₅ aliphatic. In someembodiments, R² is optionally substituted C₁₋₁₀ aliphatic. In someembodiments, R² is optionally substituted C₁₋₆ aliphatic. In someembodiments, R² is optionally substituted C₁₋₆ alkyl. In someembodiments, R² is optionally substituted hexyl, pentyl, butyl, propyl,ethyl or methyl. In some embodiments, R² is optionally substitutedhexyl. In some embodiments, R² is optionally substituted pentyl. In someembodiments, R² is optionally substituted butyl. In some embodiments, R²is optionally substituted propyl. In some embodiments, R² is optionallysubstituted ethyl. In some embodiments, R² is optionally substitutedmethyl. In some embodiments, R² is hexyl. In some embodiments, R² ispentyl. In some embodiments, R² is butyl. In some embodiments, R² ispropyl. In some embodiments, R² is ethyl. In some embodiments, R² ismethyl. In some embodiments, R² is isopropyl. In some embodiments, R² isn-propyl. In some embodiments, R² is tert-butyl. In some embodiments, R²is sec-butyl. In some embodiments, R² is n-butyl. In some embodiments,R² is benzyloxymethyl. In some embodiments, R² is benzyl.

In some embodiments, R² is optionally substituted C₁₋₂₀ heteroalkyl. Insome embodiments, R² is optionally substituted C₁₋₂₀ heteroalkyl having1-6 heteroatoms independently selected from nitrogen, sulfur, phosphorusselenium, silicon or boron. In some embodiments, R² is optionallysubstituted C₁₋₂₀ heteroalkyl having 1-6 heteroatoms independentlyselected from nitrogen, sulfur, phosphorus, selenium, silicon or boron,optionally including one or more oxidized forms of nitrogen, sulfur,phosphorus, selenium, silicon or boron.

In some embodiments, R² is —[C(R)₂]_(q)—OR. In some embodiments, R² is—CH₂OR. In some embodiments, R² is —[C(R)₂]_(q)—N(R)₂. In someembodiments, R² is —CH₂N(R)₂. In some embodiments, R² is —CH₂NHR. Insome embodiments, R² is —[C(R)₂]_(q)—SR. In some embodiments, R² is—CH₂SR. In some embodiments, R² is —[C(R)₂]_(q)—OSi(R)₃. In someembodiments, R² is —CH₂OSi(R)₃. In some embodiments, R² is—[C(R)₂]_(q)—OC(O)R. In some embodiments, R² is —CH₂OC(O)R. In someembodiments, R² is —[C(R)₂]_(q)—OC(O)OR. In some embodiments, R² is—CH₂OC(O)OR. In some embodiments, R² is —[C(R)₂]_(q)—OC(O)N(R)₂. In someembodiments, R² is —CH₂OC(O)N(R)₂. In some embodiments, R² is—CH₂OC(O)NHR. In some embodiments, R² is —[C(R)₂]_(q)—OC(O)N(R)—SO₂R. Insome embodiments, R² is —CH₂OC(O)N(R)—SO₂R. In some embodiments, R² is—CH₂OC(O)NHSO₂R. In some embodiments, R² is —[C(R)₂]_(q)—OP(OR)₂. Insome embodiments, R² is —CH₂OP(OR)₂.

Exemplary R² groups are depicted below:

In some embodiments, R¹ and R² are taken together with their interveningatoms to form an optionally substituted 4-7 membered heterocyclic ringhaving, in addition to the nitrogen atom to which R¹ is attached, 0-2heteroatoms independently selected from oxygen, nitrogen or sulfur. Insome embodiments, R¹ and R² are taken together with their interveningatoms to form an optionally substituted 4-membered heterocyclic ringhaving, in addition to the nitrogen atom to which R¹ is attached, 0-2heteroatoms independently selected from oxygen, nitrogen or sulfur. Insome embodiments, R¹ and R² are taken together with their interveningatoms to form an optionally substituted 5-membered heterocyclic ringhaving, in addition to the nitrogen atom to which R¹ is attached, 0-2heteroatoms independently selected from oxygen, nitrogen or sulfur. Insome embodiments, R¹ and R² are taken together with their interveningatoms to form an optionally substituted 6-membered heterocyclic ringhaving, in addition to the nitrogen atom to which R¹ is attached, 0-2heteroatoms independently selected from oxygen, nitrogen or sulfur. Insome embodiments, R¹ and R² are taken together with their interveningatoms to form an optionally substituted 7-membered heterocyclic ringhaving, in addition to the nitrogen atom to which R¹ is attached, 0-2heteroatoms independently selected from oxygen, nitrogen or sulfur. Insome embodiments, R¹ and R² are taken together with their interveningatoms to form

In some embodiments, R¹ and R² are taken together with their interveningatoms to form

In some embodiments, R² comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R². In some embodiments, R² comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R² comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, R² is—CH₂OH. In some embodiments, the —OH group reacts with a functionalgroup in L or M to form, for example, an ether, ester, carbamate orcarbonate ester. In some embodiments, R² reacts with a functional groupin L to form a carbonate ester. In some embodiments, R² comprises anamino group, and D is connected to L through the amino group. In someembodiments, R² comprises a —NHR group. In some embodiments, R²comprises a —NH₂ group. In some embodiments, R² is —CH₂NH₂. In someembodiments, the amino group reacts with a functional group in L or M toform, for example, an amine, imine, amide or carbamate. In someembodiments, R² comprises an —SH group, and D is connected to L throughthe —SH group. In some embodiments, the —SH group reacts with afunctional group in L or M to form, for example, a disulfide, thioetheror thioester.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a provided compound of formula II has the structureof

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

As generally defined above, each q is independently 0, 1, 2, 3 or 4. Insome embodiments, q is 0. In some embodiments, q is 1. In someembodiments, q is 2. In some embodiments, q is 3. In some embodiments, qis 4.

In some embodiments, R³ is an electron-withdrawing group. Exemplaryelectron-withdrawing groups are widely known in the art, for example,halogen, haloalkyl, carbonyl moieties (e.g., aldehyde and ketonegroups), —COOH and its derivatives (e.g., ester and amide moieties),protonated amines, quaternary ammonium groups, —CN, —NO₂, —S(O)—moieties, and —S(O)₂— moieties. In some embodiments, anelectron-withdrawing group comprises one or more —C(O)—, —C(═N—)—,—C(S)—, —S(O)—, —S(O)₂— or —P(O)— groups, and is connected to the restof a provided compound via a —C(O)—, —C(═N—)—, —C(S)—, —S(O)—, —S(O)₂—or —P(O)— group. In some embodiments, an electron-withdrawing group is—S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂,—C(O)N(R)—OR, —P(O)(R)₂, —P(O)(OR)₂, or —P(O)[N(R)₂]₂. In someembodiments, an electron-withdrawing group is halogen. In someembodiments, an electron-withdrawing group is —F. In some embodiments,an electron-withdrawing group is —Cl. In some embodiments, anelectron-withdrawing group is —Br. In some embodiments, anelectron-withdrawing group is —I. In some embodiments, anelectron-withdrawing group is —CF₃. In some embodiments, anelectron-withdrawing group is —C(O)R. In some embodiments, anelectron-withdrawing group is —C(O)OR. In some embodiments, anelectron-withdrawing group is —C(O)N(R)₂. In some embodiments, anelectron-withdrawing group is —S(O)R. In some embodiments, anelectron-withdrawing group is —S(O)₂R. In some embodiments, anelectron-withdrawing group is —S(O)₂OR. In some embodiments, anelectron-withdrawing group is —CN. In some embodiments, anelectron-withdrawing group is protonated amine. In some embodiments, anelectron-withdrawing group is —N⁺(R)₃. In some embodiments, anelectron-withdrawing group is —NO₂. In some embodiments, anelectron-withdrawing group is —P(O)(R′)₂, wherein each R′ isindependently R, —N(R)₂, —SR or —OR.

In some embodiments, R³ is an electron-withdrawing group comprising oneor more —C(O)—, —C(═N—)—, —C(S)—, —S(O)—, —S(O)₂— or —P(O)— groups, andis connected to the rest of a provided compound via a —C(O)—, —C(═N—)—,—C(S)—, —S(O)—, —S(O)₂— or —P(O)— group.

In some embodiments, R³ is an electronic-withdrawing group comprising a—S(O)₂— group, and is connected to the rest of the compound via a—S(O)₂— group. In some embodiments, R³ is —S(O)₂R, —S(O)₂—[C(R)₂]_(q)—R,—S(O)₂—[C(R)₂]_(q)—B(OR)₂, —S(O)₂—[C(R)₂]_(q)—Si(R)₃, —S(O)₂OR, or—S(O)₂N(R)₂. In some embodiments, R³ is —S(O)₂R. In some embodiments, R³is —S(O)₂R, wherein R is other than phenyl. In some embodiments, R³ is—S(O)₂—[C(R)₂]_(q)—R. In some embodiments, R³ is—S(O)₂—[C(R)₂]_(q)—B(OR)₂. In some embodiments, R³ is—S(O)₂—[C(R)₂]_(q)—Si(R)₃. In some embodiments, R³ is —S(O)₂OR. In someembodiments, R³ is —S(O)₂N(R)₂.

In some embodiments, R³ is an electronic-withdrawing group comprising a—S(O)— group, and is connected to the rest of the compound via a —S(O)—group. In some embodiments, R³ is —S(O)R.

In some embodiments, R³ is an electronic-withdrawing group comprising a—C(O)— group, and is connected to the rest of the compound via a —C(O)—group. In some embodiments, R³ is —C(O)R, —C(O)OR, —C(O)N(R)₂, or—C(O)N(R)—OR. In some embodiments, R³ is —C(O)R. In some embodiments, R³is —C(O)R, and R³ is other than —C(O)CF₃. In some embodiments, R³ is—C(O)OR. In some embodiments, R³ is —C(O)N(R)₂. In some embodiments, R³is —C(O)N(R)—OR.

In some embodiments, R³ is an electronic-withdrawing group comprising a—C(═N—)— group, and is connected to the rest of the compound via a—C(═N—)— group. In some embodiments, R³ is an electronic-withdrawinggroup comprising a —C(═NR)— group, and is connected to the rest of thecompound via a —C(═NR)— group.

In some embodiments, R³ is an electronic-withdrawing group comprising a—P(O)— group, and is connected to the rest of the compound via a —P(O)—group. In some embodiments, R³ is —P(O)(R′). In some embodiments, R³ is—P(O)(R)₂, —P(O)(OR)₂, or —P(O)[N(R)₂]₂. In some embodiments, R³ is—P(O)(R)₂. In some embodiments, R³ is —P(O)(OR)₂. In some embodiments,R³ is —P(O)[N(R)₂]₂.

In some embodiments, R³ is —S(O)₂R, —S(O)₂—[C(R)₂]_(q)—R,—S(O)₂—[C(R)₂]_(q)—B(OR)₂, —S(O)₂—[C(R)₂]_(q)—Si(R)₃, —S(O)₂OR,—S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR,—P(O)(R)₂, —P(O)(OR)₂, or —P(O)[N(R)₂]₂.

In some embodiments, R³ is other than —C(O)CF₃. In some embodiments, R³is other than tert-butyloxycarbonyl. In some embodiments, R³ is otherthan —S(O)₂Ph. In some embodiments, R³ is other than —C(O)CF₃ andtert-butyloxycarbonyl. In some embodiments, R³ is other than —S(O)₂Phand tert-butyloxycarbonyl. In some embodiments, R³ is other than—C(O)CF₃ and —S(O)₂Ph. In some embodiments, R³ is other than —S(O)₂Ph,—C(O)CF, and tert-butyloxycarbonyl. In some embodiments, R³ is otherthan —C(O)CF₃ when R¹ is —H or -Me. In some embodiments, R³ is otherthan —C(O)CF₃ when R¹ is —H. In some embodiments, R³ is other than—C(O)CF₃ when R¹ is -Me. In some embodiments, R³ is other thantert-butyloxycarbonyl when R¹ is —H or -Me. In some embodiments, R³ isother than tert-butyloxycarbonyl when R¹ is —H. In some embodiments, R³is other than tert-butyloxycarbonyl when R¹ is -Me. In some embodiments,R³ is other than —S(O)₂Ph when R¹ is —H or -Me. In some embodiments, R³is other than —S(O)₂Ph when R¹ is —H. In some embodiments, R³ is otherthan —S(O)₂Ph when R¹ is -Me. In some embodiments, R³ is other than—C(O)CF₃ and tert-butyloxycarbonyl when R¹ is —H or -Me. In someembodiments, R³ is other than —C(O)CF₃ and tert-butyloxycarbonyl when R¹is —H. In some embodiments, R³ is other than —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is -Me. In some embodiments, R³ is otherthan —C(O)CF₃ and —S(O)₂Ph when R¹ is —H or -Me. In some embodiments, R³is other than —C(O)CF₃ and —S(O)₂Ph when R¹ is —H. In some embodiments,R³ is other than —C(O)CF₃ and —S(O)₂Ph when R¹ is -Me. In someembodiments, R³ is other than —S(O)₂Ph and tert-butyloxycarbonyl when R¹is —H or -Me. In some embodiments, R³ is other than —S(O)₂Ph andtert-butyloxycarbonyl when R¹ is —H. In some embodiments, R³ is otherthan —S(O)₂Ph and tert-butyloxycarbonyl when R¹ is -Me. In someembodiments, R³ is other than —S(O)₂Ph, —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is —H or -Me. In some embodiments, R³ isother than —S(O)₂Ph, —C(O)CF₃ and tert-butyloxycarbonyl when R¹ is —H.In some embodiments, R³ is other than —S(O)₂Ph, —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is -Me.

Exemplary R³ groups include:

CO₂Et, —CHO, and —C(O)CF₃.

In some embodiments, each R^(3′) is independently R or R³. In someembodiments, each R^(3′) is independently R, —S(O)₂R,—S(O)₂—[C(R)₂]_(q)—R, —S(O)₂—[C(R)₂]_(q)—B(OR)₂,—S(O)₂—[C(R)₂]_(q)—Si(R)₃, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R,—C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —P(O)(R)₂, —P(O)(OR)₂, or—P(O)[N(R)₂]₂.

In some embodiments, R^(3′) is R. In some embodiments, R^(3′) ishydrogen. In some embodiments, R^(3′) is optionally substituted C₁₋₂₀aliphatic. In some embodiments, R^(3′) is optionally substituted C₁₋₁₅aliphatic. In some embodiments, R^(3′) is optionally substituted C₁₋₁₀aliphatic. In some embodiments, R^(3′) is optionally substituted C₁₋₆aliphatic. In some embodiments, R^(3′) is optionally substituted C₁₋₆alkyl. In some embodiments, R^(3′) is optionally substituted hexyl,pentyl, butyl, propyl, ethyl or methyl. In some embodiments, R^(3′) isoptionally substituted hexyl. In some embodiments, R^(3′) is optionallysubstituted pentyl. In some embodiments, R^(3′) is optionallysubstituted butyl. In some embodiments, R^(3′) is optionally substitutedpropyl. In some embodiments, R^(3′) is optionally substituted ethyl. Insome embodiments, R^(3′) is optionally substituted methyl. In someembodiments, R^(3′) is hexyl. In some embodiments, R^(3′) is pentyl. Insome embodiments, R^(3′) is butyl. In some embodiments, R^(3′) ispropyl. In some embodiments, R^(3′) is ethyl. In some embodiments,R^(3′) is methyl. In some embodiments, R^(3′) is isopropyl. In someembodiments, R^(3′) is n-propyl. In some embodiments, R^(3′) istert-butyl. In some embodiments, R^(3′) is sec-butyl. In someembodiments, R^(3′) is n-butyl. In some embodiments, R^(3′) isbenzyloxymethyl. In some embodiments, R^(3′) is benzyl. In someembodiments, R^(3′) is other than hydrogen.

In some embodiments, R^(3′) is optionally substituted C₁₋₂₀ heteroalkyl.In some embodiments, R^(3′) is optionally substituted C₁₋₂₀ heteroalkylhaving 1-6 heteroatoms independently selected from nitrogen, sulfur,phosphorus selenium, silicon or boron. In some embodiments, R^(3′) isoptionally substituted C₁₋₂₀ heteroalkyl having 1-6 heteroatomsindependently selected from nitrogen, sulfur, phosphorus, selenium,silicon or boron, optionally including one or more oxidized forms ofnitrogen, sulfur, phosphorus, selenium, silicon or boron.

In some embodiments, R^(3′) is R³. In some embodiments, R^(3′) is—S(O)₂R, —S(O)₂—[C(R)₂]_(q)—R, —S(O)₂—[C(R)₂]_(q)—B(OR)₂,—S(O)₂—[C(R)₂]_(q)—Si(R)₃, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R,—C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —P(O)(R)₂, —P(O)(OR)₂, or—P(O)[N(R)₂]₂. In some embodiments, R^(3′) is —S(O)₂R. In someembodiments, R^(3′) is —S(O)₂—[C(R)₂]_(q)—R. In some embodiments, R^(3′)is —S(O)₂—[C(R)₂]_(q)—B(OR)₂. In some embodiments, R^(3′) is—S(O)₂—[C(R)₂]_(q)—Si(R)₃. In some embodiments, R^(3′) is —S(O)₂OR. Insome embodiments, R^(3′) is —S(O)₂N(R)₂. In some embodiments, R^(3′) is—S(O)R. In some embodiments, R^(3′) is —C(O)R. In some embodiments,R^(3′) is —C(O)OR. In some embodiments, R^(3′) is —C(O)N(R)₂. In someembodiments, R^(3′) is —C(O)N(R)—OR. In some embodiments, R^(3′) is—P(O)(R′)₂. In some embodiments, R^(3′) is —P(O)(R)₂. In someembodiments, R^(3′) is —P(O)(OR)₂. In some embodiments, R^(3′) is—P(O)[N(R)₂].

In some embodiments, R^(3′) is other than —C(O)CF₃. In some embodiments,R^(3′) is other than tert-butyloxycarbonyl. In some embodiments, R^(3′)is other than —S(O)₂Ph. In some embodiments, R^(3′) is other than—C(O)CF₃ and tert-butyloxycarbonyl. In some embodiments, R^(3′) is otherthan —S(O)₂Ph and tert-butyloxycarbonyl. In some embodiments, R^(3′) isother than —C(O)CF₃ and —S(O)₂Ph. In some embodiments, R^(3′) is otherthan —S(O)₂Ph, —C(O)CF₃ and tert-butyloxycarbonyl. In some embodiments,R^(3′) is other than —C(O)CF₃ when R¹ is —H or -Me. In some embodiments,R^(3′) is other than —C(O)CF₃ when R¹ is —H. In some embodiments, R^(3′)is other than —C(O)CF₃ when R¹ is -Me. In some embodiments, R^(3′) isother than tert-butyloxycarbonyl when R¹ is —H or -Me. In someembodiments, R^(3′) is other than tert-butyloxycarbonyl when R¹ is —H.In some embodiments, R^(3′) is other than tert-butyloxycarbonyl when R¹is -Me. In some embodiments, R^(3′) is other than —S(O)₂Ph when R¹ is —Hor -Me. In some embodiments, R^(3′) is other than —S(O)₂Ph when R¹ is—H. In some embodiments, R^(3′) is other than —S(O)₂Ph when R¹ is -Me.In some embodiments, R^(3′) is other than —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is —H or -Me. In some embodiments, R^(3′)is other than —C(O)CF₃ and tert-butyloxycarbonyl when R¹ is —H. In someembodiments, R^(3′) is other than —C(O)CF₃ and tert-butyloxycarbonylwhen R¹ is -Me. In some embodiments, R^(3′) is other than —C(O)CF₃ and—S(O)₂Ph when R¹ is —H or -Me. In some embodiments, R^(3′) is other than—C(O)CF₃ and —S(O)₂Ph when R¹ is —H. In some embodiments, R^(3′) isother than —C(O)CF₃ and —S(O)₂Ph when R¹ is -Me. In some embodiments,R^(3′) is other than —S(O)₂Ph and tert-butyloxycarbonyl when R¹ is —H or-Me. In some embodiments, R^(3′ is other than —S(O)) ₂Ph andtert-butyloxycarbonyl when R¹ is —H. In some embodiments, R^(3′) isother than —S(O)₂Ph and tert-butyloxycarbonyl when R¹ is -Me. In someembodiments, R^(3′) is other than —S(O)₂Ph, —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is —H or -Me. In some embodiments, R^(3′)is other than —S(O)₂Ph, —C(O)CF₃ and tert-butyloxycarbonyl when R¹ is—H. In some embodiments, R³ is other than —S(O)₂Ph, —C(O)CF₃ andtert-butyloxycarbonyl when R¹ is -Me.

In some embodiments, R^(3′) comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R^(3′). In some embodiments, R^(3′) comprises an —OH, —NHR or—SH group for conjugation. In some embodiments, R^(3′) comprises an —OHgroup, and D is connected to L through the —OH group. In someembodiments, the —OH group reacts with a functional group in L or M toform, for example, an ether, ester, carbamate or carbonate ester. Insome embodiments, R^(3′) reacts with a functional group in L to form acarbonate ester. In some embodiments, R^(3′) comprises an amino group,and D is connected to L through the amino group. In some embodiments,R^(3′) comprises a —NHR group. In some embodiments, R^(3′) comprises a—NH₂ group. In some embodiments, the amino group reacts with afunctional group in L or M to form, for example, an amine, imine, amideor carbamate. In some embodiments, R^(3′) comprises an —SH group, and Dis connected to L through the —SH group. In some embodiments, the —SHgroup reacts with a functional group in L or M to form, for example, adisulfide, thioether or thioester.

In some embodiments, R⁴ is absent when

is a double bond. In some other embodiments,

is a single bond and R⁴ is R or halogen.

In some embodiments, R⁴ is R. In some embodiments, R⁴ is hydrogen. Insome embodiments, R⁴ is an optionally substituted group selected fromC₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 membered saturated orpartially unsaturated carbocyclic ring, an 8-14 membered bicyclic orpolycyclic saturated, partially unsaturated or aryl ring, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic orpolycyclic saturated or partially unsaturated heterocyclic ring having1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R⁴ is an optionally substituted C₁₋₂₀ aliphatic. Insome embodiments, R⁴ is an optionally substituted C₁₋₁₀ aliphatic. Insome embodiments, R⁴ is an optionally substituted C₁₋₆ aliphatic. Insome embodiments, R⁴ is optionally substituted C₁₋₆ alkyl. In someembodiments, R⁴ is optionally substituted hexyl, pentyl, butyl, propyl,ethyl or methyl. In some embodiments, R⁴ is optionally substitutedhexyl. In some embodiments, R⁴ is optionally substituted pentyl. In someembodiments, R⁴ is optionally substituted butyl. In some embodiments, R⁴is optionally substituted propyl. In some embodiments, R⁴ is optionallysubstituted ethyl. In some embodiments, R⁴ is optionally substitutedmethyl. In some embodiments, R⁴ is hexyl. In some embodiments, R⁴ ispentyl. In some embodiments, R⁴ is butyl. In some embodiments, R⁴ ispropyl. In some embodiments, R⁴ is ethyl. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is isopropyl. In some embodiments, R⁴ isn-propyl. In some embodiments, R⁴ is tert-butyl. In some embodiments, R⁴is sec-butyl. In some embodiments, R⁴ is n-butyl. In some embodiments,R⁴ is benzyloxymethyl. In some embodiments, R⁴ is benzyl. In someembodiments, R⁴ is an optionally substituted C₁₋₆ alkyl. In someembodiments, R⁴ is optionally substituted allyl. In some embodiments, R⁴is ally. In some embodiments, R⁴ is styrenyl. In some embodiments, R⁴ isother than hydrogen.

In some embodiments, R⁴ is optionally substituted C₁₋₂₀ heteroalkyl. Insome embodiments, R⁴ is optionally substituted C₁₋₁₀ heteroalkyl. Insome embodiments, R⁴ is optionally substituted C₁₋₆ heteroalkyl.

In some embodiments, R⁴ is optionally substituted phenyl. In someembodiments, R⁴ is substituted phenyl. In some embodiments, R⁴ isunsubstituted phenyl. In some embodiments, R⁴ is p-MeOPh.

In some embodiments, R⁴ is an optionally substituted 3-7 memberedsaturated or partially unsaturated carbocyclic ring. In someembodiments, R⁴ is an optionally substituted 3-membered saturated ring.In some embodiments, R⁴ is an optionally substituted 4-memberedsaturated or partially unsaturated carbocyclic ring. In someembodiments, R⁴ is an optionally substituted 5-membered saturated orpartially unsaturated carbocyclic ring. In some embodiments, R⁴ is anoptionally substituted 6-membered saturated or partially unsaturatedcarbocyclic ring. In some embodiments, R⁴ is an optionally substituted7-membered saturated or partially unsaturated carbocyclic ring.

In some embodiments, R⁴ is an optionally substituted 8-14 memberedbicyclic or polycyclic saturated, partially unsaturated or aryl ring. Insome embodiments, R⁴ is an optionally substituted an 8-14 memberedbicyclic or polycyclic saturated ring. In some embodiments, R⁴ is anoptionally substituted 8-14 membered bicyclic or polycyclic partiallyunsaturated ring. In some embodiments, R⁴ is an optionally substituted8-14 membered bicyclic or polycyclic aryl ring. In some embodiments, R⁴is an optionally substituted 10-membered bicyclic aryl ring. In someembodiments, R⁴ is an optionally substituted 14-membered tricyclic arylring.

In some embodiments, R⁴ is an optionally substituted 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is optionallysubstituted pyrrolyl. In some embodiments, R⁴ is optionally substitutedpyrrol-3-yl. In some embodiments, R⁴ is N-TIPS-pyrrol-3-yl. In someembodiments, R⁴ is pyrrol-3-yl.

In some embodiments, R⁴ is an optionally substituted 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 3-7 membered saturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R⁴ is an optionallysubstituted 3-7 membered partially unsaturated heterocyclic ring having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R⁴ is an optionally substituted 8-14 memberedbicyclic or polycyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁴ is an optionally substituted 8-membered bicyclic orpolycyclic heteroaryl ring having 1-5 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 8-membered bicyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 9-membered bicyclic orpolycyclic heteroaryl ring having 1-5 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 9-membered bicyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 10-membered bicyclicor polycyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 10-membered bicyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 11-membered bicyclicor polycyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 11-membered tricyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 12-membered bicyclicor polycyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 12-membered tricyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 13-membered bicyclicor polycyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 13-membered tricyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ is an optionally substituted 14-membered bicyclicor polycyclic heteroaryl ring having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ is anoptionally substituted 14-membered tricyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁴ some embodiments, R⁴ is optionally substitutedindolyl. In some embodiments, R⁴ some embodiments, R⁴ is optionallysubstituted indol-3-yl. In some embodiments, R⁴ some embodiments, R⁴ isindol-3-yl. In some embodiments, R⁴ is

In some embodiments, R⁴ is

In some embodiments, R⁴ is an optionally substituted group selected fromphenyl, a 8-14 membered bicyclic or tricyclic aryl ring, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic ortricyclic heteroaryl ring having 1-5 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In some embodiments, R⁴ is halogen. In some embodiments, R⁴ is —F. Insome embodiments, R⁴ is —Cl. In some embodiments, R⁴ is —Br. In someembodiments, R⁴ is —I.

In some embodiments, R⁴ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁴. In some embodiments, R⁴ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁴ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁴reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁴ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁴ comprises a —NHR group.In some embodiments, R⁴ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁴comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

In some embodiments, R⁵ is absent when

is a double bond.

In some embodiments, each R⁵ is independently hydrogen or an optionallysubstituted C₁₋₆ aliphatic. In some embodiments, R⁵ is hydrogen. In someembodiments, R⁵ is optionally substituted C₁₋₆ aliphatic.

In some embodiments, R⁵ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁵. In some embodiments, R⁵ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁵ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁵reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁵ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁵ comprises a —NHR group.In some embodiments, R⁵ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁵comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

As generally defined above, each of R⁶ and R^(6′) is independently R,halogen, —CN, —NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂,—N(R)S(O)₂R, or —OSi(R)₃; or R⁶ and R^(6′) are taken together to form═O, ═C(R)₂ or ═NR.

In some embodiments, each of R⁶ and R^(6′) is hydrogen. In someembodiments, each of R⁶ and R^(6′) is independently R.

In some embodiments, one of R⁶ and R^(6′) is hydrogen, and the other isR, halogen, —CN, —NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂,—N(R)S(O)₂R, or —OSi(R)₃. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is R. In some embodiments, one of R⁶ and R^(6′)is hydrogen, and the other is halogen. In some embodiments, one of R⁶and R^(6′) is hydrogen, and the other is —CN. In some embodiments, oneof R⁶ and R^(6′) is hydrogen, and the other is —NO₂. In someembodiments, one of R⁶ and R^(6′) is hydrogen, and the other is —OR. Insome embodiments, one of R⁶ and R^(6′) is hydrogen, and the other is—SR. In some embodiments, one of R⁶ and R^(6′) is hydrogen, and theother is —N(R)₂. In some embodiments, one of R⁶ and R^(6′) is hydrogen,and the other is —S(O)₂R. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is —S(O)₂N(R)₂. In some embodiments, one of R⁶and R^(6′) is hydrogen, and the other is —S(O)R. In some embodiments,one of R⁶ and R^(6′) is hydrogen, and the other is —C(O)R. In someembodiments, one of R⁶ and R^(6′) is hydrogen, and the other is —C(O)OR.In some embodiments, one of R⁶ and R^(6′) is hydrogen, and the other is—C(O)N(R)₂. In some embodiments, one of R⁶ and R^(6′) is hydrogen, andthe other is —C(O)N(R)—OR. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is —N(R)C(O)OR. In some embodiments, one of R⁶and R^(6′) is hydrogen, and the other is —N(R)C(O)N(R)₂. In someembodiments, one of R⁶ and R^(6′) is hydrogen, and the other is—N(R)S(O)₂R. In some embodiments, one of R⁶ and R^(6′) is hydrogen, andthe other is —OSi(R)₃. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is —OSi(R)₃, wherein one R is optionallysubstituted indolyl. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is —OSi(R)₃, wherein one R is optionallysubstituted indol-2-yl. In some embodiments, one of R⁶ and R^(6′) ishydrogen, and the other is —OSi(R)₃, wherein one R is optionallysubstituted.

In some embodiments, one of R⁶ and R^(6′) is hydrogen, and the other is

In some embodiments, R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂or ═NR. In some embodiments, R⁶ and R^(6′) are taken together to form═O. In some embodiments, R⁶ and R^(6′) are taken together to form═C(R)₂. In some embodiments, R⁶ and R^(6′) are taken together to form═NR.

In some embodiments, R⁶ is R. In some embodiments, R⁶ is hydrogen. Insome embodiments, R⁶ is an optionally substituted group selected fromC₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 membered saturated orpartially unsaturated carbocyclic ring, an 8-14 membered bicyclic orpolycyclic saturated, partially unsaturated or aryl ring, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic orpolycyclic saturated or partially unsaturated heterocyclic ring having1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R⁶ is halogen. In some embodiments, R⁶ is —F. Insome embodiments, R⁶ is —Cl. In some embodiments, R⁶ is —Br. In someembodiments, R⁶ is —I.

In some embodiments, R⁶ is —CN. In some embodiments, R⁶ is —NO₂. In someembodiments, R⁶ is —OR. In some embodiments, R⁶ is —SR. In someembodiments, R⁶ is —N(R)₂. In some embodiments, R⁶ is —S(O)₂R. In someembodiments, R⁶ is —S(O)₂N(R)₂. In some embodiments, R⁶ is —S(O)R. Insome embodiments, R⁶ is —C(O)R. In some embodiments, R⁶ is —C(O)OR. Insome embodiments, R⁶ is —C(O)N(R)₂. In some embodiments, R⁶ is—C(O)N(R)—OR. In some embodiments, R⁶ is —N(R)C(O)OR. In someembodiments, R⁶ is —N(R)C(O)N(R)₂. In some embodiments, R⁶ is—N(R)S(O)₂R. In some embodiments, R⁶ is —OSi(R)₃. In some embodiments,R⁶ is —OSi(R)₃, wherein one R is optionally substituted indolyl. In someembodiments, R⁶ is —OSi(R)₃, wherein one R is optionally substitutedindol-2-yl. In some embodiments, R⁶ is —OSi(R)₃, wherein one R is

In some embodiments, R⁶ is

In some embodiments, R⁶ is hydrogen, and R^(6′) is R, halogen, —CN,—NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,—C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, or—OSi(R)₃.

In some embodiments, R^(6′) is R. In some embodiments, R^(6′) ishydrogen. In some embodiments, R^(6′) is an optionally substituted groupselected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 memberedsaturated or partially unsaturated carbocyclic ring, an 8-14 memberedbicyclic or polycyclic saturated, partially unsaturated or aryl ring, a5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, a 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, a7-14 membered bicyclic or polycyclic saturated or partially unsaturatedheterocyclic ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic or polycyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R^(6′) is halogen. In some embodiments, R^(6′) is—F. In some embodiments, R^(6′) is —Cl. In some embodiments, R^(6′) is—Br. In some embodiments, R^(6′) is —I.

In some embodiments, R^(6′) is —CN. In some embodiments, R^(6′) is —NO₂.In some embodiments, R^(6′) is —OR. In some embodiments, R^(6′) is —SR.In some embodiments, R^(6′) is —N(R)₂. In some embodiments, R^(6′) is—S(O)₂R. In some embodiments, R^(6′) is —S(O)₂N(R)₂. In someembodiments, R^(6′) is —S(O)R. In some embodiments, R^(6′) is —C(O)R. Insome embodiments, R^(6′) is —C(O)OR. In some embodiments, R^(6′) is—C(O)N(R)₂. In some embodiments, R^(6′) is —C(O)N(R)—OR. In someembodiments, R^(6′) is —N(R)C(O)OR. In some embodiments, R^(6′) is—N(R)C(O)N(R)₂. In some embodiments, R^(6′) is —N(R)S(O)₂R. In someembodiments, R^(6′) is —OSi(R)₃. In some embodiments, R^(6′) is—OSi(R)₃, wherein one R is optionally substituted indolyl. In someembodiments, R^(6′) is —OSi(R)₃, wherein one R is optionally substitutedindol-2-yl. In some embodiments, R^(6′) is —OSi(R)₃, wherein one R is

In some embodiments, R^(6′) is

In some embodiments, n is 0, 1, 2, 3 or 4. In some embodiments, n is 0.In some embodiments, n is 1-4. In some embodiments, n is 1. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4.

In some embodiments, R⁶ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁶. In some embodiments, R⁶ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁶ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁶reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁶ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁶ comprises a —NHR group.In some embodiments, R⁶ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁶comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

In some embodiments, R^(6′) comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R^(6′). In some embodiments, R^(6′) comprises an —OH, —NHR or—SH group for conjugation. In some embodiments, R^(6′) comprises an —OHgroup, and D is connected to L through the —OH group. In someembodiments, the —OH group reacts with a functional group in L or M toform, for example, an ether, ester, carbamate or carbonate ester. Insome embodiments, R^(6′) reacts with a functional group in L to form acarbonate ester. In some embodiments, R^(6′) comprises an amino group,and D is connected to L through the amino group. In some embodiments,R^(6′) comprises a —NHR group. In some embodiments, R^(6′) comprises a—NH₂ group. In some embodiments, the amino group reacts with afunctional group in L or M to form, for example, an amine, imine, amideor carbamate. In some embodiments, R^(6′) comprises an —SH group, and Dis connected to L through the —SH group. In some embodiments, the —SHgroup reacts with a functional group in L or M to form, for example, adisulfide, thioether or thioester.

As generally defined above, each R⁷ is independently R, halogen, —CN,—NO₂, —OR, —OSi(R)₃, —SR, —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂,—S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR,—N(R)C(O)N(R)₂, —N(R)S(O)₂R, —P(R)₂, —P(OR)₂, —P(O)(R)₂, —P(O)(OR)₂,—P(O)[N(R)₂]₂, —B(R)₂, —B(OR)₂, or —Si(R)₃; or two R⁷ are taken togetherwith their intervening atoms to form an optionally substituted 4-7membered ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, each R⁷ isindependently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR, —N(R)₂,—S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂. —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂,—C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, —P(R)₂, —P(OR)₂,—P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂, —B(OR)₂, or —Si(R)₃. Insome embodiments, two R⁷ are taken together with their intervening atomsto form an optionally substituted 4-7 membered ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R⁷ is R. In some embodiments, R⁷ is hydrogen. Insome embodiments, R⁷ is independently hydrogen or an optionallysubstituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl,phenyl, a 3-7 membered saturated or partially unsaturated carbocyclicring, an 8-14 membered bicyclic or polycyclic saturated, partiallyunsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, a 3-7 membered saturated or partially unsaturated heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic saturated orpartially unsaturated heterocyclic ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or an 8-14membered bicyclic or polycyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R⁷ is halogen. In some embodiments, R is —F. Insome embodiments, R⁷ is —Cl. In some embodiments, R⁷ is —Br. In someembodiments, R⁷ is —I.

In some embodiments, R⁷ is —CN. In some embodiments, R⁷ is —NO₂. In someembodiments, R⁷ is —OR. In some embodiments, R⁷ is —OSi(R)₃. In someembodiments, R⁷ is —SR. In some embodiments, R⁷ is —N(R)₂. In someembodiments, R⁷ is —S(O)₂R. In some embodiments, R⁷ is —S(O)₂OR. In someembodiments, R⁷ is —S(O)₂N(R)₂. In some embodiments, R⁷ is —S(O)R. Insome embodiments, R⁷ is —C(O)R. In some embodiments, R⁷ is —C(O)OR. Insome embodiments, R⁷ is —C(O)N(R)₂. In some embodiments, R⁷ is—C(O)N(R)—OR. In some embodiments, R⁷ is —N(R)C(O)OR. In someembodiments, R⁷ is —N(R)C(O)N(R)₂. In some embodiments, R⁷ is—N(R)S(O)₂R. In some embodiments, R⁷ is —P(R)₂. In some embodiments, R⁷is —P(OR)₂. In some embodiments, R⁷ is —P(O)(R)₂. In some embodiments,R⁷ is —P(O)(OR)₂. In some embodiments, R⁷ is —P(O)[N(R)₂]₂. In someembodiments, R⁷ is —B(R)₂. In some embodiments, R⁷ is —B(OR)₂. In someembodiments, R⁷ is —Si(R)₃.

In some embodiments, R⁷ is an electron-withdrawing group. In someembodiments, R⁷ is an electron-donating group.

In some embodiments, n is 1, 2, 3 or 4, and at least one R⁷ is nothydrogen.

In some embodiments, R⁷ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁷. In some embodiments, R⁷ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁷ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁷reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁷ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁷ comprises a —NHR group.In some embodiments, R⁷ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁷comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

As generally defined above, each R⁸ is independently —(S)_(m)—R^(x)wherein m is 1-3 and R^(x) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂,—C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂, or R⁸ and R⁹ are taken togetherto form —S—, —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)S)_(p)—, —(S)_(m)—S(O)S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—. In some embodiments, each R⁸ is independently—(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR, —C(O)R, —C(O)OR,—C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂.

In some embodiments, m is 1. In some embodiments, m is 2-3. In someembodiments, m is 2. In some embodiments, m is 3.

In some embodiments, R⁸ can be converted to —(S)_(m)—H or to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)— with R⁹ when administered to a subject. Insome embodiments, R⁸ can be converted to —(S)_(m)—H or to form—(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,—(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)—(S)_(p)— with R⁹ whenadministered to a subject.

In some embodiments, R^(x) is R. In some embodiments, R⁸ is —(S)_(m)—R.In some embodiments, R⁸ is —(S)_(m)—R, wherein R⁸ can be converted to—(S)_(m)—H or to form —S—, —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—,—(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,—(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)— with R⁹ whenadministered to a subject. In some embodiments, R⁸ is —(S)_(m)—R,wherein R⁸ can be converted to —(S)_(m)—H or to form —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)— with R⁹ when administered to a subject. In someembodiments, R⁸ is —SR. In some embodiments, R⁸ is —S—S—R. In someembodiments, R⁸ is —S—S—S—R. In some embodiments, R⁸ is —(S)_(m)—R,wherein R is a cleavable group. In some embodiments, R is a cleavagegroup and when a provided compound is administered to a subject,—(S)_(m)—R is converted to —(S)_(m) ⁻. In some embodiments, R⁸ is—(S)_(m)—R, wherein R is connected to —(S)_(m)— via a carbon atom,wherein the carbon atom is substituted with a divalent substituent. Insome embodiments, R⁸ is —(S)_(m)—R, wherein R is heteroalkyl. In someembodiments, R⁸ is methyl. In some embodiments, R⁸ is CPh₃. In someembodiments, R⁸ is benzyl. In some embodiments, R⁸ is acetyl. In someembodiments, R¹ is methoxymethyl. In some embodiments, R⁸ isβ-methoxyethoxymethyl.

In some embodiments, R^(x) is —SR. In some embodiments, R^(x) is —SMe.In some embodiments, R⁸ is —(S)_(m)—SR. In some embodiments, R⁸ is—S—SR. In some embodiments, R⁸ is —S⁻S—SR. In some embodiments, R⁸ is—S—S—S—SR.

In some embodiments, R^(x) is —C(O)R. In some embodiments, R⁸ is—(S)_(m)—C(O)R. In some embodiments, R⁸ is —S—C(O)R. In someembodiments, R⁸ is —S—S—C(O)R. In some embodiments, R⁸ is —S—S—S—C(O)R.In some embodiments, R⁸ is —(S)_(m)—C(O)R, where R is methyl. In someembodiments, R⁸ is —(S)_(m)—C(O)R, where R is isopropyl.

In some embodiments, R^(x) is —C(O)OR. In some embodiments, R⁸ is—(S)_(m)—C(O)OR. In some embodiments, R⁸ is —S—C(O)OR. In someembodiments, R⁸ is —S—S—C(O)OR. In some embodiments, R⁸ is—S—S—S—C(O)OR.

In some embodiments, R^(x) is —C(O)N(R)₂. In some embodiments, R⁸ is—(S)_(m)—C(O)N(R)₂. In some embodiments, R⁸ is —S—C(O)N(R)₂. In someembodiments, R⁸ is —S—S—C(O)N(R)₂. In some embodiments, R⁸ is—S—S—S—C(O)N(R)₂.

In some embodiments, R^(x) is —C(S)R. In some embodiments, R⁸ is—(S)_(m)—C(S)R. In some embodiments, R⁸ is —S—C(S)R. In someembodiments, R⁸ is —S—S—C(S)R. In some embodiments, R⁸ is —S—S—S—C(S)R.

In some embodiments, R^(x) is —S(O)R. In some embodiments, R⁸ is—(S)_(m)—S(O)R. In some embodiments, R⁸ is —S—S(O)R. In someembodiments, R⁸ is —S—S—S(O)R. In some embodiments, R⁸ is —S—S—S—S(O)R.

In some embodiments, R^(x) is —S(O)₂R. In some embodiments, R⁸ is—(S)_(m)—S(O)₂R. In some embodiments, R⁸ is —S—S(O)₂R. In someembodiments, R⁸ is —S—S—S(O)₂R. In some embodiments, R⁸ is—S—S—S—S(O)₂R.

In some embodiments, R^(x) is —S(O)₂N(R)₂. In some embodiments, R⁸ is—(S)_(m)—S(O)₂N(R)₂. In some embodiments, R⁸ is —S—S(O)₂N(R)₂. In someembodiments, R⁸ is —S—S—S(O)₂N(R)₂. In some embodiments, R⁸ is—S—S—S—S(O)₂N(R)₂.

In some embodiments, R⁸ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁸. In some embodiments, R⁸ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁸ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁸reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁸ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁸ comprises a —NHR group.In some embodiments, R⁸ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁸comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

As generally defined above, each R⁹ is independently —(S)_(p)—R^(y)wherein p is 1-3 such that m+p is 2-4 and R^(y) is R, —SR, —C(O)R,—C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or R⁸ andR⁹ are taken together to form —(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—,—(S)_(m)C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—. In some embodiments, R⁹ is —(S)_(p)—R^(y)wherein p is 1-3 such that m+p is 2-4 and R^(y) is R, —SR, —C(O)R,—C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂. In someembodiments, R⁸ and R⁹ are taken together to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)—. In some embodiments, R⁸ and R⁹ are takentogether to form —S—, —(S)_(m)—C(R)₂—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)—. In some embodiments, R⁸ and R⁹ are takentogether to form —(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)S)_(p)—,—(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—. In some embodiments, R⁸ and R⁹ are takentogether to form —S—. In some embodiments, R⁸ and R⁹ are taken togetherto form —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—. In some embodiments, R⁸ and R⁹are taken together to form —(S)_(m)—C(R)₂—(S)_(p)—. In some embodiments,R⁸ and R⁹ are taken together to form —(S)_(m)—CH₂—(S)_(p)—. In someembodiments, R⁸ and R⁹ are taken together to form —S—CH₂—S—. In someembodiments, R⁸ and R⁹ are taken together to form —(S)_(m)—(S)_(p)—. Insome embodiments, R⁸ and R⁹ are taken together to form —S—S—. In someembodiments, R⁸ and R⁹ are taken together to form —S—S—S—. In someembodiments, R⁸ and R⁹ are taken together to form —S—S—S—S—. In someembodiments, R⁸ and R⁹ are taken together to form—(S)_(m)—C(O)—(S)_(p)—. In some embodiments, R⁸ and R⁹ are takentogether to form —S—C(O)—S—. In some embodiments, R⁸ and R⁹ are takentogether to form —(S)_(m)—C(S)—(S)_(p)—. In some embodiments, R⁸ and R⁹are taken together to form —S—C(S)—S—. In some embodiments, R⁸ and R⁹are taken together to form (S)_(m)—S(O)—(S)_(p)—. In some embodiments,R⁸ and R⁹ are taken together to form —S—S(O)—S—. In some embodiments, R⁸and R⁹ are taken together to form —(S)_(m)—S(O)₂—(S)_(p)—. In someembodiments, R⁸ and R⁹ are taken together to form —S—S(O)₂—S—.

In some embodiments, p is 1. In some embodiments, p is 2-3. In someembodiments, p is 2. In some embodiments, p is 3.

In some embodiments, m+p is 2-4. In some embodiments, m+p is 3-4. Insome embodiments, m+p is 2. In some embodiments, m+p is 3. In someembodiments, m+p is 4.

In some embodiments, R⁹ can be converted to —(S)_(p)—H or to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)— with R⁸ when administered to a subject. Insome embodiments, R⁹ can be converted to —(S)_(p)—H or to form—(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,—(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)— with R⁸ whenadministered to a subject.

In some embodiments, R^(y) is R. In some embodiments, R⁹ is —(S)_(p)—R.In some embodiments, R⁹ is —(S)_(p)—R, wherein R⁹ can be converted to—(S)_(p)—H or to form —S—, —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—,—(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—,—(S)_(m)—S(O)—(S)_(p)—, or —(S)_(m)—S(O)₂—(S)_(p)— with R⁸ whenadministered to a subject. In some embodiments, R⁹ is —(S)_(p)—R,wherein R⁹ can be converted to —(S)_(p)—H or to form —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)— with R⁸ when administered to a subject. In someembodiments, R⁹ is —SR. In some embodiments, R⁹ is —S—S—R. In someembodiments, R⁹ is —S—S—S—R. In some embodiments, R⁹ is —(S)_(p)—R,wherein R is a cleavable group. In some embodiments, R is a cleavagegroup and when a provided compound is administered to a subject,—(S)_(p)—R is converted to —(S)_(p) ⁻. In some embodiments, R⁹ is—(S)_(p)—R, wherein R is connected to —(S)_(m)— via a carbon atom,wherein the carbon atom is substituted with a divalent substituent. Insome embodiments, R⁹ is —(S)_(p)—R, wherein R is heteroalkyl. In someembodiments, R⁹ is methyl. In some embodiments, R⁹ is CPh₃. In someembodiments, R⁹ is benzyl. In some embodiments, R⁹ is acetyl. In someembodiments, R¹ is methoxymethyl. In some embodiments, R⁹ isβ-methoxyethoxymethyl.

In some embodiments, R^(y) is —SR. In some embodiments, R^(y) is —SMe.In some embodiments, R⁹ is —(S)_(p)—SR. In some embodiments, R⁹ is—S—SR. In some embodiments, R⁹ is —S⁻S—SR. In some embodiments, R⁹ is—S—S—S—SR.

In some embodiments, R^(y) is —C(O)R. In some embodiments, R⁹ is—(S)_(p)—C(O)R. In some embodiments, R⁹ is —S—C(O)R. In someembodiments, R⁹ is —S—S—C(O)R. In some embodiments, R⁹ is —S—S—S—C(O)R.In some embodiments, R⁹ is —(S)_(p)—C(O)R, where R is methyl. In someembodiments, R⁹ is —(S)_(p)—C(O)R, where R is isopropyl.

In some embodiments, R^(y) is —C(O)OR. In some embodiments, R⁹ is—(S)_(m)—C(O)OR. In some embodiments, R⁹ is —S—C(O)OR. In someembodiments, R⁹ is —S—S—C(O)OR. In some embodiments, R⁹ is—S—S—S—C(O)OR.

In some embodiments, R^(y) is —C(O)N(R)₂. In some embodiments, R⁹ is—(S)_(p)—C(O)N(R)₂. In some embodiments, R⁹ is —S—C(O)N(R)₂. In someembodiments, R⁹ is —S—S—C(O)N(R)₂. In some embodiments, R⁹ is—S—S—S—C(O)N(R)₂.

In some embodiments, R^(y) is —C(S)R. In some embodiments, R⁹ is—(S)_(p)—C(S)R. In some embodiments, R⁹ is —S—C(S)R. In someembodiments, R⁹ is —S—S—C(S)R. In some embodiments, R⁹ is —S—S—S—C(S)R.

In some embodiments, R^(y) is —S(O)R. In some embodiments, R⁹ is—(S)_(p)—S(O)R. In some embodiments, R⁹ is —S—S(O)R. In someembodiments, R⁹ is —S—S—S(O)R. In some embodiments, R⁹ is —S—S—S—S(O)R.

In some embodiments, R^(y) is —S(O)₂R. In some embodiments, R^(y) is—(S)_(p)—S(O)₂R. In some embodiments, R⁹ is —S—S(O)₂R. In someembodiments, R⁹ is —S—S—S(O)₂R. In some embodiments, R⁹ is—S—S—S—S(O)₂R.

In some embodiments, R^(y) is —S(O)₂N(R)₂. In some embodiments, R⁹ is—(S)_(p)—S(O)₂N(R)₂. In some embodiments, R⁹ is —S—S(O)₂N(R)₂. In someembodiments, R⁹ is —S—S—S(O)₂N(R)₂. In some embodiments, R⁹ is—S—S—S—S(O)₂N(R)₂.

In some embodiments, R⁸ is —(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is—SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or—S(O)₂N(R)₂; R⁹ is —(S)_(p)—R^(y) wherein p is 1-3 such that m+p is 2-4and R^(y) is —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R,or —S(O)₂N(R)₂; or R⁸ and R⁹ are taken together to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)—.

In some embodiments, R⁹ comprises an —OH, —NHR or —SH group. In someembodiments, in a provided compound of formula II, D is connected to Lthrough R⁹. In some embodiments, R⁹ comprises an —OH, —NHR or —SH groupfor conjugation. In some embodiments, R⁹ comprises an —OH group, and Dis connected to L through the —OH group. In some embodiments, the —OHgroup reacts with a functional group in L or M to form, for example, anether, ester, carbamate or carbonate ester. In some embodiments, R⁹reacts with a functional group in L to form a carbonate ester. In someembodiments, R⁹ comprises an amino group, and D is connected to Lthrough the amino group. In some embodiments, R⁹ comprises a —NHR group.In some embodiments, R⁹ comprises a —NH₂ group. In some embodiments, theamino group reacts with a functional group in L or M to form, forexample, an amine, imine, amide or carbamate. In some embodiments, R⁹comprises an —SH group, and D is connected to L through the —SH group.In some embodiments, the —SH group reacts with a functional group in Lor M to form, for example, a disulfide, thioether or thioester.

In some embodiments, neither of R⁸ and R⁹ of a provided compound is onthe α-face of the DKP. In some embodiments, R⁸ and R⁹ are on the sameface of the hexahydropyrrolo[1,2-a]pyrazine-1,4-dione moiety as N1. Insome embodiments, a provided compound is a prodrug. In some embodiments,each of R⁸ and R⁹ is independently —SR, wherein R⁸ and R⁹ can be readilyconverted into —(S)_(m)—(S)_(p)— when administered to a subject. In someembodiments, the present invention provides a method for optimizing anETP or thiodiketopiperazine compound or a derivative or an analogthereof, comprising keeping the sulfur-containing groups on the samesurface of the hexahydropyrrolo[1,2-a]pyrazine-1,4-dione moiety as N1.

As generally defined above, each D independently has the structure offormula I-c or I-d:

In some embodiments, D is a compound of formula I-c. In someembodiments, D is a compound of formula I-d. In some embodiments, D is acompound of formula I-a. In some embodiments, D is a compound of formulaI-b. When D is a compound, it is understood that said compound is bondedto L. A compound as a D unit can bond to L through any suitable atom. Insome embodiments, one or more of R¹, R², R³, R^(3′), R⁴, R⁵, R⁶, R^(6′),R⁷, R⁸ and R⁹ groups are independently linked to L. In some embodiments,R¹ is linked to L. In some embodiments, R² is linked to L. In someembodiments, R³ is linked to L. In some embodiments, R^(3′) is linked toL. In some embodiments, R⁴ is linked to L. In some embodiments, R⁵ islinked to L. In some embodiments, R⁶ is linked to L. In someembodiments, R^(6′) is linked to L. In some embodiments, R⁷ is linked toL. In some embodiments, R⁸ is linked to L. In some embodiments, R⁹ islinked to L. In some embodiments, D is linked to L through R² or R^(3′).In some embodiments, D is linked to L through R² or R³.

In some embodiments, t is 1-10. In some embodiments, t is 1-2. In someembodiments, t is 1-3. In some embodiments, t is 1-4. In someembodiments, t is 1-5. In some embodiments, t is 1-6. In someembodiments, t is 1-7. In some embodiments, t is 1-8. In someembodiments, t is 1-9. In some embodiments, t is 1-10. In someembodiments, t is 2-3. In some embodiments, t is 2-4. In someembodiments, t is 2-5. In some embodiments, t is 2-6. In someembodiments, t is 2-7. In some embodiments, t is 2-8. In someembodiments, t is 2-9. In some embodiments, t is 2-10. In someembodiments, t is 3-4. In some embodiments, t is 3-5. In someembodiments, t is 3-6. In some embodiments, t is 3-7. In someembodiments, t is 3-8. In some embodiments, t is 3-9. In someembodiments, t is 3-10. In some embodiments, t is 4-5. In someembodiments, t is 4-6. In some embodiments, t is 4-7. In someembodiments, t is 4-8. In some embodiments, t is 4-9. In someembodiments, t is 4-10. In some embodiments, t is 5-6. In someembodiments, t is 5-7. In some embodiments, t is 5-8. In someembodiments, t is 5-9. In some embodiments, t is 5-10. In someembodiments, t is 6-7. In some embodiments, t is 6-8. In someembodiments, t is 6-9. In some embodiments, t is 6-10. In someembodiments, t is 7-8. In some embodiments, t is 7-9. In someembodiments, t is 7-10. In some embodiments, t is 8-9. In someembodiments, t is 8-10. In some embodiments, t is 9-10. In someembodiments, t is 1. In some embodiments, t is 2. In some embodiments, tis 3. In some embodiments, t is 4. In some embodiments, t is 5. In someembodiments, t is 6. In some embodiments, t is 7. In some embodiments, tis 8. In some embodiments, t is 9. In some embodiments, t is 10.

In some embodiments, t is greater than 1. In some embodiments, t isgreater than one, and the D units attached to the same copy of M are thesame. In some embodiments, t is greater than one, and all the D unitsattached to the same copy of M are not the same.

In some embodiments, s is 1-10. In some embodiments, s is 1-2. In someembodiments, s is 1-3. In some embodiments, s is 1-4. In someembodiments, s is 1-5. In some embodiments, s is 1-6. In someembodiments, s is 1-7. In some embodiments, s is 1-8. In someembodiments, s is 1-9. In some embodiments, s is 1-10. In someembodiments, s is 2-3. In some embodiments, s is 2-4. In someembodiments, s is 2-5. In some embodiments, s is 2-6. In someembodiments, s is 2-7. In some embodiments, s is 2-8. In someembodiments, s is 2-9. In some embodiments, s is 2-10. In someembodiments, s is 3-4. In some embodiments, s is 3-5. In someembodiments, s is 3-6. In some embodiments, s is 3-7. In someembodiments, s is 3-8. In some embodiments, s is 3-9. In someembodiments, s is 3-10. In some embodiments, s is 4-5. In someembodiments, s is 4-6. In some embodiments, s is 4-7. In someembodiments, s is 4-8. In some embodiments, s is 4-9. In someembodiments, s is 4-10. In some embodiments, s is 5-6. In someembodiments, s is 5-7. In some embodiments, s is 5-8. In someembodiments, s is 5-9. In some embodiments, s is 5-10. In someembodiments, s is 6-7. In some embodiments, s is 6-8. In someembodiments, s is 6-9. In some embodiments, s is 6-10. In someembodiments, s is 7-8. In some embodiments, s is 7-9. In someembodiments, s is 7-10. In some embodiments, s is 8-9. In someembodiments, s is 8-10. In some embodiments, s is 9-10. In someembodiments, s is 1. In some embodiments, s is 2. In some embodiments, sis 3. In some embodiments, s is 4. In some embodiments, s is 5. In someembodiments, s is 6. In some embodiments, s is 7. In some embodiments, sis 8. In some embodiments, s is 9. In some embodiments, s is 10.

In some embodiments, s is greater than one, and the D units linked tothe same copy of L are the same. In some embodiments, s is greater thanone, and the D units linked to the same copy of L are not the same.

In some embodiments, s is smaller than 1. In some embodiments, s is 0.5.In some embodiments, a D unit is connected to two L units. In someembodiments, D has the structure of formula I-d, and is connected to twoL units. In some embodiments, D has the structure of formula I-d, andeach R² of D is independently connected to an L unit.

In some embodiments, molecules of formula II in a provided compositionhave different s or/and t values. In some embodiments, the s or/and tvalue for a composition is not an integer. In some embodiments, acomposition is relatively homogenous, and s and t have narrowerdistributions than a relatively non-homogenous composition. In someembodiments, a composition is homogenous in that the s and t values foreach molecule of formula II is the same.

In some embodiments, s=1. In some embodiments, a compound of formula IIhas the structure of formula II-c:

MLD)]_(t)   II-c.

In some embodiments, a compound of formula II has the structure offormula II-d:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula II has the structure offormula II-d:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-a-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-a-2:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-a-3:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-a-4:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-a-5:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein. In someembodiments, R² is —H or -Me; R³ is —SO₂Ph, —H or —C(O)CF₃; R⁴ is

—F or —Br; and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2 or 3,—S—C(X)—S— wherein X is O or S, —S—, —SCH₂S—, or

wherein X is —SBz, —S—C(O)R, —S—C(S)R or —S—SR. In some embodiments, R²is —H or -Me; R³ is —SO₂Ph, —H or —C(O)CF₃; R⁴ is

—F or —Br; and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2 or 3,—S—C(X)—S— wherein X is O or S, —S—, —SCH₂S—, or

wherein X is —S—C(O)R, —S—C(S)R or —S—SR. In some embodiments, R² is —Hor -Me; R³ is —SO₂Ph, —H or —C(O)CF₃; R⁴ is

—F or —Br, and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2 or 3,—S—C(X)—S— wherein X is O or S, —S—, —SCH₂S—, or

wherein X is —SAc or —S—SMe. In some embodiments, R² is —H or -Me; R³ is—SO₂Ph; R⁴ is

—F or —Br; and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2 or 3,—S—C(X)—S— wherein X is O or S, or

wherein X is —SAc or —S—SMe.

In some embodiments, a compound of formula I has the structure offormula I-b-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-b-2:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-b-3:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-b-4:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-b-5:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein. In someembodiments, R² is -Me, —CH₂OH or —CH₂OAc; R³ is —SO₂Ph, —H or —C(O)CF₃;and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2 or 3, —S—C(X)—S— whereinX is O or S, —S—, —SCH₂S— or

wherein X is —SBz, —S—C(O)R, —S—C(S)—R, -or —S—SR. In some embodiments,R² is -Me, —CH₂OH or —CH₂OAc; R³ is —SO₂Ph, —H or —C(O)CF₃; and —S_(n)—is —S—S_(n)—S— wherein n is 0, 1, 2 or 3, —S—C(X)—S— wherein X is O orS, —S—, —SCH₂S— or

wherein X is —S—C(O)R, —S—C(S)—R, -or —S—SR. In some embodiments, R² is-Me, —CH₂OH or —CH₂OAc; R³ is —SO₂Ph, —H or —C(O)CF₃; and —S_(n)— is—S—S_(n)—S— wherein n is 0, 1, 2 or 3, —S—C(X)—S— wherein X is O or S,—S—, —SCH₂S— or

wherein X is —SAc or —S—SMe. In some embodiments, R² is -Me, —CH₂OH or—CH₂OAc; R³ is —SO₂Ph; and —S_(n)— is —S—S_(n)—S— wherein n is 0, 1, 2or 3, —S—C(X)—S— wherein X is O or S, or

wherein X is —SAc or —S—SMe.

In some embodiments, D has the structure of formula I-c-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, D has the structure of formula I-c-2:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, D has the structure of formula I-c-3:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-c-4:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, D has the structure of formula I-d-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-d-2:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-d-3:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I has the structure offormula I-d-4:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, D has the structure of formula I-a-1. In someembodiments, D has the structure of formula I-a-2. In some embodiments,D has the structure of formula I-a-3. In some embodiments, D has thestructure of formula I-a-4. In some embodiments, D has the structure offormula I-a-5. In some embodiments, D has the structure of formulaI-b-1. In some embodiments, D has the structure of formula I-b-2. Insome embodiments, D has the structure of formula I-b-3. In someembodiments, D has the structure of formula I-b-4. In some embodiments,D has the structure of formula I-b-5.

In some embodiments, M is a cell-specific ligand unit. Exemplarycell-specific ligand unit are widely known in the art, including thosedescribed herein.

In some embodiments, M includes within its scope any unit of a Ligand(L^(L)) that, for example, binds or reactively associates or complexeswith a receptor, antigen or other receptive moiety associated with agiven target-cell population. In some embodiments, a Ligand is amolecule that binds to, complexes with, or reacts with a moiety of acell population sought to be therapeutically or otherwise biologicallymodified. In some embodiments, the Ligand unit acts to deliver the drugunit (e.g., a compound of formulae I-c or I-d) to the particular targetcell population with which the Ligand unit reacts. Such Ligands include,but are not limited to, large molecular weight proteins such as, forexample, full-length antibodies, antibody fragments, smaller molecularweight proteins, polypeptide or peptides, lectins, glycoproteins,non-peptides, vitamins, nutrient-transport molecules (such as, but notlimited to, transferring, or any other cell binding molecule orsubstance), nucleic acids and their derivatives and analogs. In someembodiments, a Ligand is modified from its corresponding natural formby, for example, amino acid mutations (including substitutions,deletions, insertions, etc), incorporation of unnatural building blocks(such as unnatural amino acids and unnatural nucleotides (e.g., thosewith unnatural bases, sugars, and/or internucleoside linkages)),chemical modifications, and/or conjugation with other small or macromolecules. In some embodiments, M comprises a reactive functional groupsuch as an amine (—NH₂), aldehyde (—CHO), carboxyl (—COOH) or asulfhydryl group (—SH), or can be modified to contain such a functionalgroup. In some embodiments, M is coupled to the linker moiety of theconjugate by way of a free reactive sulfhydryl (—SH), amine (—NH₂),aldehyde (—CHO), ketone or carboxyl (—COOH) group or can be modified tocontain such a sulfhydryl, amine, aldehyde, ketone or carboxyl group. Insome embodiments, M is an antibody (Ab). In some embodiments, L is acovalent bond and M is directly connected to D. In some embodiments, Lis not a covalent bond and M is connected to D through L.

In some embodiments, a Ligand unit can form a bond to a linker unit (L)via a carbon atom of the Ligand. In some embodiments, a Ligand unit canform a bond to a linker unit (L) via a heteroatom of the Ligand.Heteroatoms that may be present on a Ligand unit include sulfur (in someembodiments, from a sulfhydryl group of a Ligand), oxygen (in someembodiments, from a carbonyl, carboxyl or hydroxyl group of a Ligand)and nitrogen (in some embodiment, from a primary or secondary aminogroup of a Ligand). These heteroatoms can be present on the Ligand inthe Ligand's natural state, for example a naturally-occurring antibody,or can be introduced into the Ligand via chemical modification.

In some embodiments, a Ligand has a sulfhydryl group and the Ligandbonds to the Linker unit via the sulfhydryl group's sulfur atom.

In yet some other embodiments, the Ligand has one or more lysineresidues that can be chemically modified to introduce one or moresulfhydryl groups. The Ligand unit bonds to the Linker unit via thesulfhydryl group's sulfur atom. The reagents that can be used to modifylysines include, but are not limited to, N-succinimidylS-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut'sReagent).

In some embodiments, the Ligand can have one or more carbohydrate groupsthat can be chemically modified to have one or more sulfhydryl groups.The Ligand unit bonds to the Linker Unit, such as the Stretcher Unit,via the sulfhydryl group's sulfur atom.

In some embodiments, the Ligand can have one or more carbohydrate groupsthat can be oxidized to provide an aldehyde (—CHO) group (see, for e.g.,Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The correspondingaldehyde can form a bond with a Reactive Site on a Stretcher. Reactivesites on a Stretcher that can react with a carbonyl group on a Ligandinclude, but are not limited to, hydrazine and hydroxylamine. Otherprotocols for the modification of proteins for the attachment orassociation to a compound of formulae I-c or I-d are described inColigan et al., Current Protocols in Protein Science, vol. 2, John Wiley& Sons (2002).

Useful non-immunoreactive protein, polypeptide, or peptide Ligandsinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, lectins and apoprotein from lowdensity lipoprotein.

In some embodiments, M is an antibody. In some embodiments, an antibodyrefers to any immunoglobulin, whether natural or wholly or partiallysynthetically produced. All derivatives thereof which maintain specificbinding ability are also included. In some embodiments, an antibody alsocovers any protein having a binding domain which is homologous orlargely homologous to an immunoglobulin-binding domain. Such proteinsmay be derived from natural sources, or partly or wholly syntheticallyproduced. An antibody may be monoclonal or polyclonal. An antibody maybe a member of any immunoglobulin class, including any of the humanclasses: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, anantibody may be a member of the IgG immunoglobulin class. In someembodiments, M is an antibody fragment or characteristic portion of anantibody, which refers to any derivative of an antibody which is lessthan full-length. In general, an antibody fragment retains at least asignificant portion of the full-length antibody's specific bindingability. Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, scFv, Fv, dsFv diabody, and Fd fragments. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antibody fragment maycomprise multiple chains which are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amultimolecular complex. In some embodiments, a functional antibodyfragment comprises at least about 50 amino acids. In some embodiments, afunctional antibody fragment comprises at least about 200 amino acids.In some embodiments, an antibody may be a human antibody. In someembodiments, an antibody may be a humanized antibody. Exemplary antibodyembodiments for M include intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody fragments, solong as they exhibit the desired biological activity.

Useful polyclonal antibodies as Ligand are, in some embodiments,heterogeneous populations of antibody molecules derived from the sera ofimmunized animals. Various procedures well known in the art may be usedfor the production of polyclonal antibodies to an antigen-of-interest.For example, for the production of polyclonal antibodies, various hostanimals can be immunized by injection with an antigen of interest orderivative thereof, including but not limited to rabbits, mice, rats,and guinea pigs. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete) adjuvant, mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

Useful monoclonal antibodies as Ligand are, in some embodiments,homogeneous populations of antibodies to a particular antigenicdeterminant (e.g., a cancer cell antigen, a viral antigen, a microbialantigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid,or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Köhler and Milstein(1975, Nature 256, 495-497), the human B cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, and IgD and anysubclass thereof. The hybridoma producing the mAbs of use in thisinvention may be cultivated in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. USA. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79;and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Similar procedures are disclosed in InternationalPublication No. WO 93/08829, and in Traunecker et al., EMBO J.10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. Nucleic acids with sequences encoding the immunoglobulin heavychain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In an embodiment of this approach, the bispecific antibodies have ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation(International Publication No. WO 94/04690).

For further details for generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 1986, 121:210; Rodrigueset al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992,Bio/Technology 10: 163-167; Carter et al., 1995, J. of Hematotherapy4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Usingsuch techniques, bispecific antibodies can be prepared for use in thetreatment or prevention of disease as defined herein.

Bifunctional antibodies are also described, in European PatentPublication No. EPA 0 105 360. In some embodiments, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies and/or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example, in International Publication WO83/03679, and European Patent Publication No. EPA 0 217 577.Bifunctional antibodies include those biologically prepared from a“polydoma” or “quadroma” or which are synthetically prepared withcross-linking agents such as bis-(maleimido)-methyl ether (“BMME”), orwith other cross-linking agents familiar to those skilled in the art.

An antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to cancer cell antigens,viral antigens, or microbial antigens or other antibodies bound to tumorcells or matrix. In this regard, “functionally active” means that thefragment, derivative or analog is able to elicit anti-anti-idiotypeantibodies that recognize the same antigen that the antibody from whichthe fragment, derivative or analog is derived recognized. Specifically,in an exemplary embodiment the antigenicity of the idiotype of theimmunoglobulin molecule can be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognizes the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart (e.g., the BIA core assay) (See, for e.g., Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md.; Kabat E et al., 1980, J. ofImmunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, which contain the variable region, thelight chain constant region and the CHI domain of the heavy chain can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Other useful antibodies are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-54), or any other molecule with the same specificity as theantibody.

In some embodiments, an antibody is an immunoglobulin antibody. In someembodiments, an immunoglobulin antibody recognizes a tumor-associatedantigen.

In some embodiments, an antibody is a recombinant antibody, such as achimeric and/or a humanized monoclonal antibodies, comprising both humanand non-human portions, which can be made using standard recombinant DNAtechniques. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and humanimmunoglobulin constant regions. See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397. Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.See, e.g., Queen, U.S. Pat. No. 5,585,089. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in InternationalPublication No. WO 87/02671; European Patent Publication No. 184,187;European Patent Publication No. 171496; European Patent Publication No.173494; International Publication No. WO 86/01533; U.S. Pat. No.4,816,567; European Patent Publication No. 12,023; Berter et al, 1988,Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; andShaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985,Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060.

In some embodiments, completely human antibodies are desirable and canbe produced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806. Other human antibodies canbe obtained commercially from, for example, Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al. (1994) Biotechnology12:899-903). Human antibodies can also be produced using varioustechniques known in the art, including phage display libraries(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991); Quan, M. P, and Carter, P. 2002. The rise ofmonoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease,Jardieu, P. M, and Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y.,Chapter 20, pp. 427-469).

In other embodiments, an antibody is a fusion protein of an antibody, ora functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond) at, forexample, either the N-terminus or the C-terminus, to an amino acidsequence of another protein (or portion thereof, preferably at least 10,20 or 50 amino acid portion of the protein) that is not the antibody. Insome embodiments, the antibody or fragment thereof is covalently linkedto the other protein at the N-terminus of the constant domain. In someembodiments, an antibody is a fusion protein comprising analbumin-binding peptide sequence (ABP).

In some embodiments, an antibody is conjugated to one or more polymerunits. In some embodiments, a polymer unit is functionalized so that itis used for connection with -L-. In some embodiments, a polymer unit isfunctionalized in such a way that a controlled number of -L- and drugunits (e.g., compounds of formula I-c or I-d) can be linked, thereforecontrolling the amount of a provided compound conjugated to an antibodymolecule.

In some embodiments, antibodies include analogs and derivatives that aremodified, e.g., by the covalent attachment of any type of molecule, bychemically modifying one or more amino acid residues, and/or byintroducing one or more mutations, including substitutions, insertionsand deletions, as long as such modifications permits the antibody toretain its antigen binding immunospecificity. For example, but not byway of limitation, the derivatives and analogs of the antibodies includethose that have been further modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular antibody unit or other protein, amino acid mutations, etc. Anyof numerous chemical modifications can be carried out by knowntechniques, including, but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis in the presence oftunicamycin, etc.

In some embodiments, an analog or derivative of an antibody, peptide orprotein contains one or more unnatural amino acid residues. In someembodiments, an antibody, peptide or protein contains a controllednumber of unnatural amino acid residues, which are used for conjugationto -L- and a provided compound, e.g., a compound of formula I-c or I-d,therefore controlling the number of copies of a provided compoundconjugated to the antibody, peptide or protein molecule. In someembodiments, an antibody contains one unnatural amino acid residue forconjugation. In some embodiments, an antibody contains two unnaturalamino acid residues for conjugation. In some embodiments, an antibodycontains three unnatural amino acid residues for conjugation. In someembodiments, an antibody contains four unnatural amino acid residues forconjugation. In some embodiments, an antibody contains five unnaturalamino acid residues for conjugation. In some embodiments, an antibodycontains six unnatural amino acid residues for conjugation. In someembodiments, an antibody contains seven unnatural amino acid residuesfor conjugation. In some embodiments, an antibody contains eightunnatural amino acid residues for conjugation. In some embodiments, anantibody contains nine unnatural amino acid residues for conjugation. Insome embodiments, an antibody contains ten unnatural amino acid residuesfor conjugation. Suitable unnatural amino acid residues and methods fortheir incorporation are widely known and practiced in the art. In someembodiments, an unnatural amino acid residue comprises an optionallysubstituted alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl or heterocyclyl group that is not found in natural amino acidresidues. In some embodiments, an unnatural amino acid comprises atleast one —N₃ group. In some embodiments, an unnatural amino acidcomprises at least one alkenyl group. In some embodiments, an unnaturalamino acid comprises at least one alkynyl group. In some embodiments, anunnatural amino acid comprises at least one ketone group. In someembodiments, an unnatural amino acid comprises at least one aldehydegroup. In some embodiments, an unnatural amino acid residue is ap-acetylphenylalanine residue. In some embodiments, an unnatural aminoacid residue is a formylglycine residue. Methods for conjugation througha —N₃, alkenyl, alkynyl, ketone and/or aldehyde are widely known andpracticed in the art, including but not limited to click chemistry,metathesis and/or reactions with amines, alkoxyamines and hydrazides.Among other things, conjugation through functionalized unnatural aminoacid residues provides control over both the number and placement of aprovide compound (site-specific conjugation).

In some embodiments, M comprises at least one aldehyde or ketone group.In some embodiments, M comprises at least one aldehyde group. In someembodiments, M is coupled to the rest of a provided compound viareaction of the aldehyde or ketone group with an amine (e.g., reductiveamination), a substituted hydrazine (e.g., forming a hydrazone), ahydrazide, or an alkoxyamine (e.g., forming oximes). In someembodiments, a coupling reaction comprises the use of N-substitutedalkoxyamine, and an intramolecular transformation similar to aPictet-Spengler reaction, for example:

In some embodiments, an aldehyde group of M, or another type of reactivegroup of M that is used for linkage to L, is converted from anotherfunctional group. In some embodiments, an another functional group iswithin the side chain of a natural amino acid residue or an incorporatedunnatural amino acid through, for example, a chemical or enzymatictransformation. In some embodiments, a transformation is an enzymatictransformation. In some embodiments, a transformation coverts a cysteineresidue into a formylglycine residue. In some embodiments, such atransformation is promoted by a formylglycine-generation enzyme.Exemplary antibodies and the preparation and use thereof are describedin US Patent Application Publication US 2010/0210543 and US2012/0183566.

In some embodiments, an antibody is a cysteine engineered antibody. Insome embodiments, a cysteine engineered antibody comprises one or morefree cysteine amino acid, which is a cysteine amino acid residue whichhas been engineered into a parent antibody, has a thiol functional group(—SH), and is not paired as an intermolecular or intramoleculardisulfide bridge. In some embodiments, a cysteine engineered antibodycomprising a free cysteine amino acid having a thiol reactivity value inthe range of 0.6 to 1.0 as defined in U.S. Pat. No. 7,855,275. Exemplarycysteine engineered antibodies are widely described in the art,including but not limited to those described in U.S. Pat. Nos. 7,855,275and 7,521,541, the entirety of each of which is incorporated byreference herein. In some embodiments, a cysteine engineered antibody issite-specifically and efficiently coupled with a thiol-reactive reagent,such as a multifunctional linker reagent or a drug-linker intermediate(e.g., -L-(D)s, wherein D is a compound of I-c or I-d).

In some embodiments, M is a diabody, tribody, tetrabody, minibody ornanobody.

The antibodies include antibodies having modifications (e.g.,substitutions, deletions or additions) in amino acid residues thatinteract with Fc receptors. In particular, antibodies include antibodieshaving modifications in amino acid residues identified as involved inthe interaction between the anti-Fc domain and the FcRn receptor (see,e.g., International Publication No. WO 97/34631. Antibodiesimmunospecific for a cancer cell antigen can be obtained commercially,for example, from Genentech (San Francisco, Calif.) or produced by anymethod known to one of skill in the art such as, e.g., chemicalsynthesis or recombinant expression techniques. The nucleotide sequenceencoding antibodies immunospecific for a cancer cell antigen can beobtained, e.g., from the GenBank database or a database like it, theliterature publications, or by routine cloning and sequencing.

In some embodiments, known antibodies for the treatment or prevention ofcancer can be used. Antibodies immunospecific for a cancer cell antigencan be obtained commercially or produced by any method known to one ofskill in the art such as, e.g., recombinant expression techniques. Thenucleotide sequence encoding antibodies immunospecific for a cancer cellantigen can be obtained, e.g., from the GenBank database, or a databaselike it, the literature publications, or by routine cloning andsequencing. Examples of antibodies available for the treatment of cancerinclude, but are not limited to, humanized anti-HER2 monoclonalantibody, HERCEPTIN® (trastuzumab; Genentech) for the treatment ofpatients with metastatic breast cancer. RITUXAN® (rituximab; Genentech)which is a chimeric anti-CD20 monoclonal antibody for the treatment ofpatients with non-Hodgkin's lymphoma: OvaRex (AltaRex Corporation, MA)which is a murine antibody for the treatment of ovarian cancer; Panorex(Glaxo Wellcome, NC) which is a murine IgG_(2a) antibody for thetreatment of colorectal cancer, Cetuximab Erbitux (Imclone Systems Inc.,NY) which is an anti-EGFR IgG chimeric antibody for the treatment ofepidermal growth factor positive cancers, such as head and neck cancer,Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for thetreatment of sarcoma; Campath I/H (Leukosite, MA) which is a humanizedIgG, antibody for the treatment of chronic lymphocytic leukemia (CLL);Smart MI95 (Protein Design Labs, Inc., CA) which is a humanizedanti-CD33 IgG antibody for the treatment of acute myeloid leukemia(AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanizedanti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma;Smart ID10 (Protein Design Labs, Inc., CA) which is a humanizedanti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma;Oncolym (Techniclone, Inc., CA) which is a radiolabeled murineanti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma;Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for thetreatment of Hodgkin's Disease or non-Hodgkin's lymphoma; Avastin(Genentech, Inc., CA) which is an anti-VEGF humanized antibody for thetreatment of lung and colorectal cancers; Epratuzamab (Immunomedics,Inc., NJ and Amgen, Calif.) which is an anti-CD22 antibody for thetreatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ)which is a humanized anti-CEA antibody for the treatment of colorectalcancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens: CA125 (ovarian),CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostatespecific antigen (prostate), prostatic acid phosphatase (prostate),epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA(colorectal), gp100 (melanoma), MARTI (melanoma), PSA (prostate), IL-2receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma),CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionicgonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma),mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogeneproduct (carcinomas). Some specific, useful antibodies include, but arenot limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J.,Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A.,Hellstrom, I., Hellstrom, K. E., “Cure of Xenografted Human Carcinomasby BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64(Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J.,Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza,A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom,K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability,Potency, and Efficacy of Carcinoma-reactive BR64-DoxorubicinImmunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against theCD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L.,Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and invivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res.2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb and2F2 mAb, and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A.,Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functionaleffects of CD30 on a large granular lymphoma cell line YT” J. Immunol.,151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res. 62(13):3736-42).Many other internalizing antibodies that bind to tumor associatedantigens can be used and have been reviewed (Franke, A. E., Sievers, E.L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy ofacute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000, 15,459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: acoming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel,S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

In certain embodiments, the antibody is Trastuzumab (full length,humanized anti-HER2 (MW 145167)), HerceptinF(ab′)₂ (derived fromanti-HER2 enzymatically (MW 100000)), 4D5 (full-length, murine antiHER2,from hybridoma), rhu4D5 (transiently expressed, full-length humanizedantibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8(full-length, murine antiHER2, with mutated FcRn binding domain), or Hg(“Hingeless” full-length humanized 4D5, with heavy chain hinge cysteinesmutated to serines. Expressed in E. coli (therefore non-glycosylated)).In certain embodiments, the antibody is not Trastuzumab (full length,humanized anti-HER2 (MW 145167)), HerceptinF(ab′)₂ (derived fromanti-HER2 enzymatically (MW 100000)), 4D5 (full-length, murine antiHER2,from hybridoma), rhu4D5 (transiently expressed, full-length humanizedantibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8(full-length, murine antiHER2, with mutated FcRn binding domain), or Hg(“Hingeless” full-length humanized 4D5, with heavy chain hinge cysteinesmutated to serines. Expressed in E. coli (therefore non-glycosylated)).

In another specific embodiment, known antibodies for the treatment orprevention of an autoimmune disease are used in accordance with thecompositions and methods of the invention. Antibodies immunospecific foran antigen of a cell that is responsible for producing autoimmuneantibodies can be obtained from any organization (e.g., a university ora company) or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.In another embodiment, useful antibodies are immunospecific for thetreatment of autoimmune diseases include, but are not limited to,Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, Anti-CardiolipinAntibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SMAntibody; Anti-Mitochondrial Antibody; Thyroid Antibody; MicrosomalAntibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo; Anti-U₁RNP;Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital Cells Antibody;Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti centromere;Anti-Fibrillarin, and Anti-GBM Antibody. In some embodiments, anantibody is an anti-AGS 16, anti-AGS5, anti-Nectin 4, anti-CA9,anti0mesothelin, anti-Cripto, anti-CD138, anti-CD70, anti-GPNMB,anti-CD56, anti-alpha integrin, anti-CD22, anti-PSMA, anti-Her2,anti-CD19, anti-DS6, anti-CD30, or anti-CD70 antibody.

In certain embodiments, useful antibodies can bind to a receptor or areceptor complex expressed on an activated lymphocyte. The receptor orreceptor complex can comprise an immunoglobulin gene superfamily member,a TNF receptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin, or acomplement control protein. Non-limiting examples of suitableimmunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22,CD28, CD79, CD90, CD52/CTLA-4, PD-1, and ICOS. Non-limiting examples ofsuitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c,CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, andCD104. Non-limiting examples of suitable lectins are C-type, S-type, andI-type lectin.

In some embodiments, the Ligand binds to an activated lymphocyte that isassociated with an autoimmune disease.

In some embodiments, useful Ligands immunospecific for a viral or amicrobial antigen are monoclonal antibodies. The antibodies may bechimeric, humanized or human monoclonal antibodies. As used herein, theterm “viral antigen” includes, but is not limited to, any viral peptide,polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein,influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax,herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) andhepatitis B surface antigen) that is capable of eliciting an immuneresponse. As used herein, the term “microbial antigen” includes, but isnot limited to, any microbial peptide, polypeptide, protein, saccharide,polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenicprotozoa, or yeast polypeptide including, e.g., LPS and capsularpolysaccharide 5/8) that is capable of eliciting an immune response.

Antibodies immunospecific for a viral or microbial antigen can beobtained commercially, for example, from BD Biosciences (San Francisco,Calif.), Chemicon International, Inc. (Temecula, Calif.), or VectorLaboratories, Inc. (Burlingame, Calif.) or produced by any method knownto one of skill in the art such as, e.g., chemical synthesis orrecombinant expression techniques. The nucleotide sequence encodingantibodies that are immunospecific for a viral or microbial antigen canbe obtained, e.g., from the GenBank database or a database like it,literature publications, or by routine cloning and sequencing.

In a specific embodiment, useful Ligands are those that are useful forthe treatment or prevention of viral or microbial infection inaccordance with the methods disclosed herein. Examples of antibodiesavailable useful for the treatment of viral infection or microbialinfection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD)which is a humanized anti-respiratory syncytial virus (RSV) monoclonalantibody useful for the treatment of patients with RSV infection; PRO542(Progenics) which is a CD4 fusion antibody useful for the treatment ofHIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; PROTOVIR(Protein Design Labs, Inc., CA) which is a humanized IgG1 antibodyuseful for the treatment of cytomegalovirus (CMV); and anti-LPSantibodies.

Other antibodies useful in the treatment of infectious diseases include,but are not limited to, antibodies against the antigens from pathogenicstrains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria meningitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridiunm perfringens,Clostridium tetani, Henophilus influenzae, Kiebsiella pneumoniae,Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus,Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneunmocystiscarinii, Francisella tularensis, Brucella abortus, Brucella suis,Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis,Aspergillus fimnigatus, Candida albicans, Blastomyces dermatitidis,Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoebahistolytica. Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,Trichomonas vaginalis, Trvoanosoma gambiense, Trypanosoma rhodesiense,Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmaniabraziliensis, Pneumocstis pneumonia, Plasmodium vivax, Plasnmodiumlalciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis,Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis,Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni,Schistosoma haematobium, and hookworms).

Other antibodies useful in this invention for treatment of viral diseaseinclude, but are not limited to, antibodies against antigens ofpathogenic viruses, including as examples and not by limitation:Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae,Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenzaviruses, mumps, measles, respiratory syncytial virus, rubella,Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitisvirus, Rhinoviridae, Coronaviridae, Rotoviridae, and HumanImmunodeficiency Virus.

Transmembrane or otherwise tumor-associated polypeptides that arespecifically expressed on the surface of one or more particular type(s)of cancer cell as compared to on one or more normal non-cancerouscell(s) were identified for cancer diagnosis and therapy. In someembodiments, such tumor-associated polypeptides are more abundantlyexpressed on the surface of the cancer cells as compared to on thesurface of the non-cancerous cells. The identification of suchtumor-associated cell surface antigen polypeptides has given rise to theability to specifically target cancer cells for destruction viaantibody-based therapies.

Antibodies as embodiments for M include, but are not limited to,antibodies against tumor-associated antigens (TAA). Suchtumor-associated antigens are known in the art, and can be prepared foruse in generating antibodies using methods and information which arewell known in the art. Often, such tumor-associated polypeptides aremore abundantly expressed on the surface of the cancer cells as comparedto on the surface of the non-cancerous cells. The identification of suchtumor-associated cell surface antigen polypeptides has given rise to theability to specifically target cancer cells for destruction viaantibody-based therapies. Examples of TAA include (1)-(36), but are notlimited to TAA (1)-(36) listed below. Tumor-associated antigens targetedby antibodies include all amino acid sequence variants and isoformspossessing at least about 70%, 80%, 85%, 90%, or 95% sequence identityrelative to the sequences identified in the corresponding sequenceslisted (1-36), or the sequences identified in the cited references, orwhich exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. In some embodiments, TAA having amino acid sequence variantsexhibit substantially the same biological properties or characteristicsas a TAA having the sequence found in the corresponding sequences below.For example, a TAA having a variant sequence generally is able to bindspecifically to an antibody that binds specifically to the TAA with thecorresponding sequence listed. The sequences and specifically recitedherein are expressly incorporated by reference.

In some embodiments, an antibody binds to an ErbB receptor, and/or oneor more of receptors (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203);

(2) E16 (LAT1, SLC7A5);

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4) 0772P (CA125, MUC16);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter3b);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B);

(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene);

(9) ETBR (Endothelin type B receptor);

(10) MSG783 (RNF124, hypothetical protein FLJ20315);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor);

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792);

(15) CD79b (CD79B, CD790, IGb (immunoglobulin-associated beta), B29);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein Ia), SPAP1B, SPAP1C);

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20Rα;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3;

(27) CD22 (B-cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with IgM molecules, transduces a signalinvolved in B-cell differentiation);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia);

(30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) thatbinds peptides and presents them to CD4+ T lymphocytes);

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis);

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation);

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); and

(36) TENB2 (putative transmembrane proteoglycan, related to theEGF/heregulin family of growth factors and follistatin).

Other exemplary monoclonal antibodies which may recognize tumorassociated antigen include:

Antigen Site Monoclonal Recognized Antibodies Reference Lung TumorsKS1/4 N. M. Varki, et al., Cancer Res. 44: 681, 1984 534, F8; F.Cuttitta, et al., in: G. L. Wright 604A9 (ed) Monoclonal Antibodies andCancer, Marcel Dekker, Inc., NY., p. 161, 1984. Squamous Lung G1, LuCa2,Kyoizumi et al., Cancer Res., LuCa3, LuCa4 45: 3274, 1985. Small CellLung TFS-2 Okabe et al., Cancer Res. Cancer 45: 1930, 1985. Colon Cancer11.285.14 G. Rowland, et al., Cancer 14.95.55 Immunol.Immunother., 19:1, 1985 NS-3a-22, NS- Z. Steplewski, et al., Cancer 10 Res., 41: 2723,1981. NS-19-9, NS- 33a NS-52a, 17- 1A Erbitux ® Carcinoembryonic MoAb 35or Acolla, R. S. et al., Proc. ZCE025 Natl. Acad. Sci., (USA), 77: 563,1980. Melanoma 9.2.27 T. F. Bumol and R. A. Reisfeld, Proc. Natl. Acad.Sci., (USA), 79: 1245, 1982. p97 96.5 K. E. Hellstrom, et al.,Monoclonal Antibodies and Cancer, loc. cit. p. 31. Antigen T65 T101Boehringer-Mannheim, P.O. Box 50816, Indianapolis, IN 46250 FerritinAntiferrin Boehringer-Mannheim, P.O. Box 50816, Indianapolis, IN 46250R24 W. G. Dippold, et al., Proc. Natl. Acad. Sci. (USA), 77: 6114, 1980Neuroblastoma P1 153/3 R. H. Kennet and F. Gilbert, Science, 203: 1120,1979. MIN 1 J. T. Kemshead in Monoclonal Antibodies and Cancer, loc.cit. p. 49. UJ13A Goldman et al., Pediatrics, 105: 252, 1984. GliomaBF7, GE2, N. de Tribolet, et al., in CG12 Monoclonal Antibodies andCancer, loc. cit. p. 81 Ganglioside L6 I. Hellstrom et al. Proc. Natl.Acad. Sci. (U.S.A) 83: 7059 (1986); U.S. Pat. Nos. 4,906,562, issuedMar. 6, 1990 and 4,935,495, issued Jun. 19, 1990. Chimeric L6 U.S. Ser.No. 07/923,244, (abandoned) filed Oct. 27, 1986, equivalent to PCTPatent Publication, WO 88/03145, published May 5, 1988. Lewis Y BR64U.S. Ser. Nos. 07/289,635 (abandoned) filed Dec. 22, 1988, and U.S. Ser.No. 07/443,696 (now U.S. Pat. No. 5,242,824) Nov. 29, 1989, equivalentto European Patent Publication, EP A 0 375 562, published Jun. 27, 1990,fucosylated BR96, U.S. Ser. Nos. 07/374,947 Chimeric (abandoned) filedJun. 30, 1989, Lewis Y BR96 and U.S. Ser. No. 07/544,246 (abandoned)filed Jun, 26, 1990, equivalent to PCT Patent Publication, WO 91/00295,published Jan. 10, 1991. Breast Cancer B6.2, B72.3 D. Colcher, et al.,in Monoclonal Antibodies and Cancer, loc. cit. p. 121. Herceptin ®Baselga, et al., J. Clin. Oncol., 14: 737-744, 1996; U.S. Pat. No.5,821,337 Mylotarg ® Osteogenic 791T/48, M. J. Embleton, ibid, p. 181Sarcoma 791T/36 Sarcoma 791T/36 Leukemia CALL 2 C. T. Teng, et al.,Lancet, 1: 01, 1982 anti-idiotype R. A. Miller, et al., N. Eng. J. Med.,306: 517, 1982 Ovarian Cancer OC 125 R. C. Bast, et. al., J. Clin.Invest., 68: 1331, 1981. Prostrate Cancer D83.21, P6.2, J. J. Starling,et al., in Turp-27 Monoclonal Antibodies and Cancer, loc. cit., p. 253Renal Cancer A6H, D5D P. H. Lange, et al., Surgery, 98: 143, 1985.Non-Hodgkins Rituxan ® lymphoma

In some embodiments, an antibody binds to one or more of the antigenslisted below, or one or more of the antigens expressed by the tumorand/or tumor types listed below:

Antigen category Examples of antigens Tumor types expressing antigenCluster of differentiation CD20 non-Hodgkin lymphoma (CD) antigens CD30Hodgkin lymphoma CD33 Acute myelogenous leukemia CD52 Chroniclymphocytic leukemia Glycoproteins EpCAM Epithelial tumors (breast,colon, lung) CEA Epithelial tumors (breast, colon, lung) gpA33Colorectal carcinoma Mucins Epithelial tumors (breast, colon, lung,ovarian) TAG-72 Epithelial tumors (breast, colon, lung) Carbonicanhydrase IX Renal cell carcinoma PSMA Prostate carcinoma Folate bindingprotein Ovarian tumors Glycolipids Gangliosides (e.g., GD2, GD3, GM2)Neuroectodermaltumors, some epithelial tumors Carbohydrates Lewis-Y²Epithelial tumors (breast, colon, lung, prostate) Vascular targets VEGFTumor vasculature VEGFR Epithelium-derived solid tumors αVβ3 Tumorvasculature α5β1 Tumor vasculature Growth factors ErbB1/EGFR Glioma,lung, breast, colon, head and neck tumors ErbB2/HER2 Breast, colon,lung, ovarian, prostate tumors ErbB3 Breast, colon, lung, ovarian,prostate tumors c-MET Epithelial tumors (breast, ovary, lung) IGF1RLung, breast, head and neck, prostate, thyroid, glioma EphA3 Lung,kidney, colon, melanoma, glioma, hematological malignancies TRAIL-R1,TRAIL-R2 Solid tumors (colon, lung, pancreas) and hematologicalmalignancies RANKL Prostate cancer and bone metastases Stromal andextracellular FAP Epithelial tumors (colon, breast, lung, head and neck,pancreas) matrix antigens Tenascin Glioma, epithelial tumors (breast,prostate)

Exemplary tumor-associated antigens and specific antibodies thereto werealso included in WO04/045516; WO03/000113; WO02/016429; WO02/16581;WO03/024392; WO04/016225; WO01/40309; and U.S. Provisional patentapplication Ser. No. 60/520,842.

Exemplary antibodies also include: Herceptin® (trastuzumab)=full length,humanized antiHER2 (MW 145167), Herceptin F(ab′)2=derived from antiHER2enzymatically (MW 100000), 4D5=full-length, murine antiHER2, fromhybridoma, rhu4D5=transiently expressed, full-length humanized antibody,rhuFab4D5=recombinant humanized Fab (MW 47738), 4D5Fc8=full-length,murine antiHER2, with mutated FcRn binding domain.

In some embodiments, an antibody specifically binds to a receptorencoded by an ErbB gene. In some embodiments, an antibody bindsspecifically to an ErbB receptor selected from EGFR, HER2, HER3 andHER4. In some embodiments, an antibody specifically binds to anextracellular domain of the HER2 receptor and inhibits the growth oftumor cells which overexpress HER2 receptor. In some embodiments,HERCEPTIN® (trastuzumab) selectively binds to the extracellular domain(ECD) of the human epidermal growth factor receptor 2 protein, HER2(ErbB2) (U.S. Pat. Nos. 5,821,337; 6,054,297; 6,407,213; 6,639,055;Coussens et al (1985) Science 230:1132-9; Slamon, et al (1989) Science244:707-12).

In some embodiments, an antibody is a monoclonal antibody, e.g. a murinemonoclonal antibody, a chimeric antibody, or a humanized antibody. Insome embodiments, a humanized antibody is huMAb4D5-1, huMAb4D5-2,huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8(Trastuzumab). The antibody may be an antibody fragment, e.g. a Fabfragment. In some embodiments, an antibody is hu4D5Fabv8.

As generally defined above, each L is independently a linker unit. Insome embodiments, L is a covalent bond. In some embodiments, a linkerunit is a chemical moiety comprising a covalent bond or a chain of atomsthat covalently attaches an antibody to a drug moiety (D). In someembodiments, L is an optionally substituted bivalent C₁₋₅₀ aliphatic orheteroalkylene group, wherein one or more carbon atoms, optionally withone or more hydrogen atoms attached thereto, are optionally andindependently replaced by —O—, ═O, —N(R)—, ═N—,

≡N, —S—, ═S, —S(O)—, —S(O)₂—, —S(O)₂N(R)—, —C(O)—, —OC(O)—, —OC(O)O—,—C(S)—, —C(O)N(R)—, —N(R)C(O)O—, N(R)C(O)N(R)—,

—Se—, —Se(O)—,

an amino acid residue, or -Cy¹-, wherein each -Cy¹- is independently:

-   -   a bivalent optionally substituted monocyclic ring independently        selected from phenylene, a 3-8 membered saturated or partially        unsaturated carbocyclylene, a 5-6 membered heteroarylene having        1-4 heteroatoms independently selected from nitrogen, oxygen, or        sulfur, or a 3-8 membered saturated or partially unsaturated        heterocyclylene having 1-4 heteroatoms independently selected        from nitrogen, oxygen, or sulfur, or    -   a bivalent optionally substituted bicyclic ring independently        selected from an 8-10 membered arylene, a 7-10 membered        saturated or partially unsaturated carbocyclylene, an 8-10        membered heteroarylene having 1-5 heteroatoms independently        selected from nitrogen, oxygen, or sulfur, or a 7-10 membered        saturated or partially unsaturated heterocyclylene having 1-5        heteroatoms selected from nitrogen, oxygen, or sulfur, or    -   a bivalent optionally substituted tricyclic ring independently        selected from 14 membered arylene, a 9-20 membered saturated or        partially unsaturated carbocyclylene, a 9-14 membered        heteroarylene having 1-8 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, or a 9-20 membered saturated or        partially unsaturated heterocyclylene having 1-8 heteroatoms        independently selected from nitrogen, oxygen, or sulfur; or    -   a bivalent optionally substituted tetracyclic ring independently        selected from a 16-18 membered arylene, an 11-30 membered        saturated or partially unsaturated carbocyclylene, a 15-18        membered heteroarylene having 1-8 heteroatoms independently        selected from nitrogen, oxygen, or sulfur, or an 11-30 membered        saturated or partially unsaturated heterocyclylene having 1-12        heteroatoms independently selected from nitrogen, oxygen, or        sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedmonocyclic ring.

In certain embodiments, -Cy¹- is bivalent optionally substitutedphenylene.

In certain embodiments, -Cy¹- is a bivalent optionally substituted 3-8membered saturated carbocyclylene. In certain embodiments, -Cy¹- is abivalent optionally substituted 3-8 membered partially unsaturatedcarbocyclylene. In certain embodiments, -Cy¹- is a bivalent optionallysubstituted 5-6 membered saturated carbocyclylene. In certainembodiments, -Cy¹- is a bivalent optionally substituted 5-6 memberedpartially unsaturated carbocyclylene.

In certain embodiments, -Cy¹- is a bivalent optionally substituted 5membered heteroarylene having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is abivalent optionally substituted 5 membered heteroarylene having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Incertain embodiments, -Cy¹- is a bivalent optionally substituted 6membered heteroarylene having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is abivalent optionally substituted 6 membered heteroarylene having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, -Cy¹- is a bivalent optionally substituted 3-8membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹-is a bivalent optionally substituted 3-8 membered saturatedheterocyclylene having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is a bivalentoptionally substituted 5-6 membered saturated heterocyclylene having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Incertain embodiments, -Cy¹- is a bivalent optionally substituted 5-6membered saturated heterocyclylene having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹-is a bivalent optionally substituted 5 membered saturatedheterocyclylene having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is a bivalentoptionally substituted 6 membered saturated heterocyclylene having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, -Cy¹- is a bivalent optionally substituted 3-8membered partially unsaturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted 3-8 memberedpartially unsaturated heterocyclylene having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted 5-6 memberedpartially unsaturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted 5-6 memberedpartially unsaturated heterocyclylene having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted 5 memberedpartially unsaturated heterocyclylene having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted 6 memberedpartially unsaturated heterocyclylene having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is bivalent optionally substitutednaphthylene.

In some embodiments, -Cy¹- is a bivalent optionally substituted bicyclic7-10 membered saturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted bicyclic 7 membered saturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted bicyclic 8 membered saturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted bicyclic 9membered saturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted bicyclic 10 membered saturatedcarbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substituted bicyclic7-10 membered partially unsaturated carbocyclylene. In some embodiments,-Cy¹- is a bivalent optionally substituted bicyclic 7 membered partiallyunsaturated carbocyclylene. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 8 membered partially unsaturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted bicyclic 9 membered partially unsaturated carbocyclylene. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 10membered partially unsaturated carbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substituted bicyclic8-10 membered heteroarylene having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 8-10 membered heteroarylenehaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedbicyclic 8 membered heteroarylene having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 8 membered heteroarylenehaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedbicyclic 9 membered heteroarylene having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 9 membered heteroarylenehaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedbicyclic 10 membered heteroarylene having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 10 membered heteroarylenehaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In some embodiments, -Cy¹- is a bivalent optionally substituted bicyclic7-10 membered saturated heterocyclylene having 1-5 heteroatoms selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted bicyclic 7-10 membered saturatedheterocyclylene having 1-2 heteroatoms selected from nitrogen, oxygen,or sulfur. In some embodiments, -Cy¹- is a bivalent optionallysubstituted bicyclic 7 membered saturated heterocyclylene having 1-5heteroatoms selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted bicyclic 7membered saturated heterocyclylene having 1-2 heteroatoms selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 8 membered saturated heterocyclylenehaving 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 8membered saturated heterocyclylene having 1-2 heteroatoms selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 9 membered saturated heterocyclylenehaving 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 9membered saturated heterocyclylene having 1-2 heteroatoms selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 10 membered saturated heterocyclylenehaving 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 10membered saturated heterocyclylene having 1-2 heteroatoms selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substituted bicyclic7-10 membered partially unsaturated heterocyclylene having 1-5heteroatoms selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted bicyclic 7-10membered partially unsaturated heterocyclylene having 1-2 heteroatomsselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 7 membered partiallyunsaturated heterocyclylene having 1-5 heteroatoms selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 7 membered partially unsaturatedheterocyclylene having 1-2 heteroatoms selected from nitrogen, oxygen,or sulfur. In some embodiments, -Cy¹- is a bivalent optionallysubstituted bicyclic 8 membered partially unsaturated heterocyclylenehaving 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 8membered partially unsaturated heterocyclylene having 1-2 heteroatomsselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted bicyclic 9 membered partiallyunsaturated heterocyclylene having 1-5 heteroatoms selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted bicyclic 9 membered partially unsaturatedheterocyclylene having 1-2 heteroatoms selected from nitrogen, oxygen,or sulfur. In some embodiments, -Cy¹- is a bivalent optionallysubstituted bicyclic 10 membered partially unsaturated heterocyclylenehaving 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted bicyclic 10membered partially unsaturated heterocyclylene having 1-2 heteroatomsselected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14 membered arylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-20 membered saturated carbocyclylene. In some embodiments,-Cy¹- is a bivalent optionally substituted tricyclic 10-20 memberedsaturated carbocyclylene. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-20 membered saturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted tricyclic 12-18 membered saturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-14membered saturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 14-16 membered saturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted tricyclic 16-18 membered saturated carbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-20 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 10-20membered partially unsaturated carbocyclylene. In some embodiments,-Cy¹- is a bivalent optionally substituted tricyclic 12-20 memberedpartially unsaturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 12-18 membered partiallyunsaturated carbocyclylene. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-14 membered partially unsaturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted tricyclic 14-16 membered partially unsaturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted tricyclic 16-18 membered partially unsaturatedcarbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-14 membered heteroarylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 9-14membered heteroarylene having 1-6 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 9-14 membered heteroarylenehaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-14 membered heteroarylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 9-14membered heteroarylene having 2-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 9-14 membered heteroarylenehaving 4-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 10-14 membered heteroarylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 10-14membered heteroarylene having 1-6 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 10-14 membered heteroarylenehaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 10-14 membered heteroarylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 10-14membered heteroarylene having 2-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 10-14 membered heteroarylenehaving 4-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered heteroarylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-14membered heteroarylene having 1-6 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 12-14 membered heteroarylenehaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered heteroarylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-14membered heteroarylene having 2-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is abivalent optionally substituted tricyclic 12-14 membered heteroarylenehaving 4-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-20 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 9-20membered saturated heterocyclylene having 1-6 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 9-20 membered saturatedheterocyclylene having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 9-20 membered saturated heterocyclylenehaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-20 membered saturated heterocyclylene having 2-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 9-20membered saturated heterocyclylene having 4-6 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered saturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic12-20 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-20membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 12-20 membered saturatedheterocyclylene having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-20 membered saturatedheterocyclylene having 2-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-20 membered saturatedheterocyclylene having 4-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered saturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic12-18 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-18membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 12-18 membered saturatedheterocyclylene having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-18 membered saturatedheterocyclylene having 2-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-18 membered saturatedheterocyclylene having 4-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered saturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic12-14 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 12-14membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 12-14 membered saturatedheterocyclylene having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-14 membered saturatedheterocyclylene having 2-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 12-14 membered saturatedheterocyclylene having 4-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered saturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic14-16 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 14-16membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 14-16 membered saturatedheterocyclylene having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 14-16 membered saturatedheterocyclylene having 2-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 14-16 membered saturatedheterocyclylene having 4-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered saturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic16-18 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tricyclic 16-18membered saturated heterocyclylene having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tricyclic 16-18 membered saturatedheterocyclylene having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 16-18 membered saturatedheterocyclylene having 2-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tricyclic 16-18 membered saturatedheterocyclylene having 4-6 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 9-20 membered partially unsaturated heterocyclylene having 1-8heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic9-20 membered partially unsaturated heterocyclylene having 1-6heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic9-20 membered partially unsaturated heterocyclylene having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic9-20 membered partially unsaturated heterocyclylene having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic9-20 membered partially unsaturated heterocyclylene having 2-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tricyclic9-20 membered partially unsaturated heterocyclylene having 4-6heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having2-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-20 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having2-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-18 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having2-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 12-14 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having2-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 14-16 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having2-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtricyclic 16-18 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 16-18 membered arylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered saturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted tetracyclic 12-24 membered saturatedcarbocyclylene. In some embodiments, -Cy¹- is a bivalent optionallysubstituted tetracyclic 14-24 membered saturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated carbocyclylene. In some embodiments, -Cy¹- is abivalent optionally substituted tetracyclic 20-24 membered saturatedcarbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic14-24 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered partially unsaturated carbocyclylene. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered partially unsaturated carbocyclylene.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-18 membered heteroarylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-18 membered heteroarylene having 1-6 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- isa bivalent optionally substituted tetracyclic 15-18 memberedheteroarylene having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, -Cy¹- is a bivalentoptionally substituted tetracyclic 15-18 membered heteroarylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-18 membered heteroarylene having 2-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-18 membered heteroarylene having 4-8 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-18 membered heteroarylene having 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic 15membered heteroarylene having 3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is a bivalenttetracyclic 15 membered heteroarylene having 3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein -Cy¹- is substitutedwith at least two R groups. In certain embodiments, -Cy¹- is a bivalenttetracyclic 15 membered heteroarylene having 3 heteroatoms independentlyselected from oxygen and nitrogen, wherein -Cy¹- is substituted with atleast two R groups, and wherein the at least two R groups are onadjacent atoms and are taken together with their intervening atoms toform an optionally substituted 3-8 membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In certain embodiments, -Cy¹- is abivalent tetracyclic 15 membered heteroarylene having 3 heteroatomsindependently selected from oxygen or nitrogen, wherein -Cy¹- issubstituted with at least two R groups, and wherein the at least two Rgroups are on adjacent atoms and are taken together with theirintervening atoms to form an optionally substituted 8 membered partiallyunsaturated ring having 1 heteroatom independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, the one heteroatomof the above described optionally substituted 8 membered partiallyunsaturated ring is nitrogen.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered saturated heterocyclylene having 1-12heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 1-10 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 4-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 4-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-30 membered saturated heterocyclylene having 6-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered saturated heterocyclylene having 1-12heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 1-10 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 4-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 4-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic11-25 membered saturated heterocyclylene having 6-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered saturated heterocyclylene having 1-12heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 1-10 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 4-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 4-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic12-24 membered saturated heterocyclylene having 6-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered saturated heterocyclylene having 1-12heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 1-10 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 4-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 4-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic15-20 membered saturated heterocyclylene having 6-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered saturated heterocyclylene having 1-12heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 1-10 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 1-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 1-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 4-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 4-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, -Cy¹- is a bivalent optionally substituted tetracyclic20-24 membered saturated heterocyclylene having 6-8 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-12 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-10 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having4-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-30 membered partially unsaturated heterocyclylene having6-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-12 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-10 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having4-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 11-25 membered partially unsaturated heterocyclylene having6-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-12 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-10 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having4-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 12-24 membered partially unsaturated heterocyclylene having6-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-12 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-10 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having4-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 15-20 membered partially unsaturated heterocyclylene having6-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-12 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-10 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having4-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having4-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In some embodiments, -Cy¹- is a bivalent optionally substitutedtetracyclic 20-24 membered partially unsaturated heterocyclylene having6-8 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, L is a covalent bond.

In some embodiments, a linker units include a divalent radical such asan alkyldiyl, an arylene, a heteroarylene, moieties such as:—[C(R)₂]_(n)O[C(R)₂]_(n)—, repeating units of alkyloxy (e.g.polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.polyethyleneamino, Jeffamine™); and diacid ester and amides includingsuccinate, succinamide, diglycolate, malonate, and caproamide.

In some embodiments, a linker unit (or linker, L) is a bifunctional ormultifunctional moiety which can be used to link one or more Drugmoieties (D) and an M unit, such as an antibody unit (Ab) to formantibody-drug conjugates (ADC) of formula II. Antibody-drug conjugates(ADC) can be conveniently prepared using a linker unit having reactivefunctionality for binding to the Drug and to the Antibody. In someembodiments, a cysteine thiol of M (e.g., a cysteine engineered antibody(Ab)), or a functional group of a modified or unnatural amino acidresidue of M (e.g., an antibody), can form a bond with a functionalgroup of a linker reagent, a drug moiety or drug-linker intermediate.

In some embodiments, a linker unit has a reactive site which has anelectrophilic group that is reactive to a nucleophilic group of M. Insome embodiments, a linker unit has a reactive site which has anelectrophilic group that is reactive to a nucleophilic cysteine presenton an antibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a Linker and forms a covalent bond to a Linker.In some embodiments, a nucleophilic group is an amino group. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups.

In some embodiments, a linker unit has a reactive site which has anucleophilic group that is reactive to an electrophilic group of M. Insome embodiments, an electrophilic group of M is an aldehyde or ketonegroup. As described herein, in some embodiments, an aldehyde or ketonegroup is incorporated through, for example, inclusion of unnatural aminoacids and/or chemical modifications (e.g., modification of an amino acidside, change, oxidation of carbohydrates of glycosylated proteins, etc).

In some embodiments, a linker unit L has the structure of:

-A_(a)-W_(w)—Y_(y)—

wherein:-A- is a Stretcher unit covalently attached to M;a is 0 or 1;each —W— is independently an Amino Acid unit;w is independently an integer ranging from 0 to 12;—Y— is a Spacer unit covalently attached to the drug moiety; andy is 0, 1 or 2.

Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking an M unit,such as an antibody, to an amino acid unit (—W—) or a drug unit (D). Insome embodiments, M has a nucleophilic group that forms a bond with anelectrophilic functional group of a Stretcher unit. In some embodiments,M is an antibody, and a nucleophilic group, such as an amino or thiolgroup, forms a bond with an electrophilic functional group of aStretcher unit. In some embodiments, M has an electrophilic group thatforms a bond with a nucleophilic functional group of a Stretcher unit.In some embodiments, M is an antibody, and an electrophilic group, suchas an aldehyde or ketone group, forms a bond with a nucleophilicfunctional group of a Stretcher unit, such as a thiol, amino,hydroxylamine, hydrazine or hydrazide group. In some embodiments, anucleophilic group is —SH. In some embodiments, a nucleophilic group is—N(R)₂. In some embodiments, a nucleophilic group is a hydroxylaminegroup. In some embodiments, a nucleophilic group is a hydrazine group.In some embodiments, a nucleophilic group is a hydrazide group. In someembodiments, an electrophilic group is a ketone group. In someembodiments, an electrophilic group is an aldehyde group. In someembodiments, an electrophilic group comprises an unsaturated bond, suchas a carbon-carbon double bond, a carbon-carbon triple bond, or acarbonyl group. In some embodiments, an electrophilic group is anunsaturated carbon-carbon bond conjugated to an electron-withdrawinggroup. In some embodiments, an electrophilic group is a carbon-carbondouble bond conjugated to an electron-withdrawing group.

In some embodiments, a compound of formula II has the structure offormula L-IIIa or L-IIIb. In some embodiments, exemplary Stretcher unitsinclude those depicted in ADCs having the structure of Formulae L-IIIaand L-IIIb, wherein Ab is an antibody, —W—, —Y—, D, w and y are asdefined above and described herein, and R¹⁷ is an optionally substituteddivalent radical selected from —(CH₂)_(r)—, C₃-C₈ carbocyclyl,—O—(CH₂)_(r)—, arylene, —(CH₂)_(r)-arylene, -arylene-(CH₂)_(r)—,—(CH₂)_(r-3)—C₈ carbocyclyl)-, —(C₃-C₈ carbocyclyl)-(CH₂)_(r)—, C₃-C₈heterocyclyl, —(CH₂)_(r)—(C₃-C₈ heterocyclyl)-, —(C₃-C₈heterocyclyl)-(CH₂)_(r)—, —(CH₂)_(r)C(O)NR^(b)(CH₂)_(r)—,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)CH₂—, and—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂)_(r)—; wherein R^(b) is H, C₁-C₆ alkyl,phenyl, or benzyl; and r is independently an integer ranging from 1-10.

In some embodiments, arylene includes divalent aromatic hydrocarbonradicals of 6-20 carbon atoms derived by the removal of two hydrogenatoms from a parent aromatic ring system. Typical arylene groupsinclude, but are not limited to, radicals derived from benzene,substituted benzene, naphthalene, anthracene, biphenyl, and the like.

In some embodiments, heterocyclyl groups include a ring system in whichone or more ring atoms is a heteroatom, e.g. nitrogen, oxygen, andsulfur. In some embodiments, a heterocyclic radical comprises 1 to 20carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Insome embodiments, a heterocyclic group may be an optionally substitutedmonocyclic group having 3 to 7 ring members (2 to 6 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S) or a bicyclic grouphaving 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatomsselected from N, O, P, and S), for example: a bicyclo[4,5], [5,5],[5,6], or [6,6] system. In some embodiments, a heterocyclic group isderived from an exemplary heterocyclic compounds described in Paquette,Leo A.; “Principles of Modem Heterocyclic Chemistry” (W. A. Benjamin,New York, 1968) (for example, Chapters 1, 3, 4, 6, 7, and 9); “TheChemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley& Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocyclic groups include by way of example and notlimitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl),thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl,pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl,tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl,quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, 4Ah-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl,acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl.

In some embodiments, carbocyclyl groups include a saturated orunsaturated ring having 3 to 7 carbon atoms as a monocyclic or 7 to 12carbon atoms as a bicyclic group. In some embodiments, monocycliccarbocyclic groups have 3 to 6 ring atoms, or 5 or 6 ring atoms. In someembodiments, bicyclic carbocyclic groups have 7 to 12 ring atoms, e.g.arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples ofmonocyclic carbocyclic groups include cyclopropyl, cyclobutyl,cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl,cyclohexyl, I-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cycloheptyl, and cyclooctyl.

In some embodiments, a Stretcher unit is that of formula L-IIIa, and isderived from maleimido-caproyl (MC) wherein R¹⁷ is —(CH₂)₅—:

In some embodiments, a Stretcher unit is that of formula L-IIIa, and isderived from maleimido-propanoyl (MP) wherein R¹⁷ is —(CH₂)₂—:

In some embodiments, a Stretcher unit is that of formula L-IIIa whereinR¹⁷ is —(CH₂CH₂O)_(r)—CH₂— and r is 2:

In some embodiments, a Stretcher unit is that of formula L-IIIa whereinR¹⁷ is —(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— wherein R^(b) is H andeach r is 2:

In some embodiments, a Stretcher unit is that of formula L-IIIb whereinR¹⁷ is —(CH₂)₅—:

In some other embodiments, a Stretcher unit is linked to M, e.g., anAntibody unit via a disulfide bond between a sulfur atom of an Antibodyunit and a sulfur atom of the Stretcher unit. An exemplary Stretcherunit is depicted in a compound of formula II having the structure offormula L-IV, wherein R, Ab-, —W—, —Y—, -D, w and y are as defined aboveand described herein.

Ab-SS—R⁷—C(O)—W_(w)—Y_(y)-D)_(t)  L-IV

In some embodiments, the reactive group of the Stretcher contains athiol-reactive functional group that can form a bond with a freecysteine thiol of an antibody. Examples of thiol-reaction functionalgroups include, but are not limited to, maleimide, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Exemplary Stretcher units are depicted in a compound of formula IIhaving the structure of formula L-Va and L-Vb, wherein R, Ab-, —W—, —Y—,-D, w and y are as defined above and described herein.

AbS—C(O)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(t)  L-Va

AbS—C(S)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(t)  L-Vb

In some embodiments, a linker unit is a dendritic type or polymericlinker for covalent attachment of more than one drug moiety through abranching, multifunctional linker moiety to an antibody (for example,Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215;Sun et at (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King(2002) Tetrahedron Letters 43:1987-1990:). In some embodiments, a linkerunit is dendritic. In some embodiments, a linker unit is polymeric. Insome embodiments, dendritic or polymeric linkers can increase the molarratio of drug to antibody, i.e. loading, which is related to the potencyof the ADC. In some embodiments, a dendritic or polymeric linker unitprovides favorable conditions for conjugation of a drug unit and alinker unit. In some embodiments, a drug unit is first coupled to adendritic or polymeric linker unit, followed by coupling of the linkerunit with M. In some embodiments, a first coupling between a drug unitand a dendritic or polymeric linker unit can be conducted in organicsolvents or mixtures thereof that may not be friendly to an M unit suchas a protein (e.g., an antibody).

Exemplary dendritic linker reagents are depicted below. In someembodiments, up to nine nucleophilic drug moiety reagents can beconjugated by reaction with the chloroethyl nitrogen mustard functionalgroups. It is understood that the number of drug moiety reagents thatcan be conjugated can be adjusted by varying the number of X, Y or Zgroups.

In some embodiments, a Spacer unit comprises branched, self-immolative2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenoldendrimer units (WO 2004/01993; Szalai et al (2003) J. Amer. Chem. Soc.125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126:1726-1731;Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499).

In some embodiments, all Drug units (D) connected to a dendritic orpolymeric linker or Spacer unit are the same. In some embodiments, allDrug units (D) connected to a dendritic or polymeric linker or Spacerunit are not the same. In some embodiments, two or more types of drugunits (D) are connected to the same copy of a dendritic or polymericlinker or Spacer unit.

Amino Acid Unit

In some embodiments, a linker unit comprises amino acid residues. Insome embodiments, the Amino Acid unit (—W_(w)—), when present, links thedrug units (D) to M, for example, an antibody, optionally through one ormore Stretcher unit.

In some embodiments, —W_(w)— is a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit. Exemplary suitableamino acid residues for W include those occurring naturally, as well asnon-naturally occurring amino acid analogs, such as citrulline. In someembodiments, each —W— unit independently has the structure of:

wherein w is an integer ranging from 0 to 12, R¹⁹ is -L¹⁹-R^(L), L¹⁹ isan optionally substituted C₀₋₆ bivalent aliphatic chain orheteroalkylene, and R^(L) is hydrogen or an optionally substituted groupselected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 memberedsaturated or partially unsaturated carbocyclic ring, an 8-14 memberedbicyclic or polycyclic saturated, partially unsaturated or aryl ring, a5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, a 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, a7-14 membered bicyclic or polycyclic saturated or partially unsaturatedheterocyclic ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic or polycyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹⁹ is hydrogen,methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

In some embodiments, an Amino Acid unit can be enzymatically cleaved byone or more enzymes, including a tumor-associated protease, to liberatea Drug moiety (D), or an active unit comprising the Drug and part of theLinker unit (L).

Useful —W_(w)-units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzymes, for example, atumor-associated protease. In some embodiments, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

Exemplary —W_(w)— Amino Acid units include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).

When R¹⁹ is other than hydrogen, the carbon atom to which R¹⁹ isattached is chiral. In some embodiments, each carbon atom to which R¹⁹is attached is independently in the (S) or (R) configuration, or aracemic mixture. In some embodiments, the amino acid units arestereochemically pure. In some embodiments, the amino acid units areenantiomerically pure. In some embodiments, the amino acid unites areracemic. In some embodiments, the amino acid units arediastereomerically pure. In some embodiments, the amino acid unitscontain a plurality of stereoisomers. In some embodiments, the aminoacid units contain a plurality of enantiomers. In some embodiments, theamino acid units contain a plurality of multiple diastereomers. In someembodiments, the amino acid units contain a plurality of stereoisomers,wherein the amount of each of the stereoisomers is pre-determined.

Spacer Unit

In some embodiments, the Spacer units (—Y_(y)—), when present (y=1 or2), link an Amino Acid unit (—W—) to the drug moiety (D) when an AminoAcid unit is present (w=1-12). In some embodiments, the Spacer unitslink the Stretcher unit to the Drug moiety when the Amino Acid unit isabsent. In some embodiments, the Spacer units link the drug moiety tothe antibody unit when both the Amino Acid unit and Stretcher unit areabsent (w, y=0). In some embodiments, a Spacer unit is of two generaltypes: self-immolative and non self-immolative. In some embodiments, aSpacer unit is self-immolative. In some embodiments, a Spacer unit isnon self-immolative. In some embodiments, an ADC compound has a nonself-immolative Spacer unit, and part or all of the Spacer unit remainsbound to the Drug moiety after cleavage, for example enzymatic cleavage,of the linker unit (L). When an ADC containing a glycine-glycine Spacerunit or a glycine Spacer unit undergoes enzymatic cleavage via atumor-cell associated-protease, a cancer-cell-associated protease or alymphocyte-associated protease, a glycine-glycine-Drug moiety or aglycine-Drug moiety is cleaved from the ADC. In some embodiments, anindependent hydrolysis reaction takes place within the target cell,cleaving the glycine-Drug moiety bond and liberating the Drug.

In some embodiments, —Y_(y)— is a p-aminobenzylcarbamoyl (PAB) unitwhose phenylene portion is substituted with Q_(m′) wherein Q isoptionally substituted —C₁-C₈ alkyl, optionally substituted —O—(C₁-C₈alkyl), -halogen, -nitro or —CN; and m′ is an integer ranging from 0-4.

Exemplary non self-immolative Spacer units (—Y—) include -Gly-Gly-,-Gly-, -Ala-Phe-, and -Val-Cit-.

In some embodiments, y is 0 and L has no space unit.

In some embodiments, an ADC containing a self-immolative Spacer unit canrelease D. In one embodiment, —Y— is a PAB group that is linked to—W_(w)— via the amino nitrogen atom of the PAB group, and connecteddirectly to D via a carbonate, carbamate or ether group, wherein acompound of formula II has the structure of:

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al (1995) Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al(1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at glycine (Kingsbury et al(1984) J. Med. Chem. 27:1447) are also examples of self-immolativespacer useful in ADCs.

In some embodiments, a Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS), which can be used to incorporate andrelease multiple drugs, having the structure:

comprising a 2-(4-aminobenzylidene)propane-1,3-diol dendrimer unit (WO2004/043493; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494),wherein n is 0 or 1. In some embodiments, t is 0-4.

In some embodiments, the Spacer units (—Y_(y)—) are selected fromformula (L-X)-(L-XII):

Exemplary ADCs of formula II include those depicted in formula L-XIIIa(val-cit), L-XIIIb (MC-val-cit), and L-XIIIc (MC-val-cit-PAB):

Other exemplary ADCs of formula II include:

wherein X is: —CH₂-1,4-cyclohexylene-, —(CH₂)_(n′)—, —(CH₂CH₂O)_(n′)—,—CH₂-1,4-cyclohexylene-C(O)—N(R)—(CH₂)_(n′)—, phenylene,-phenylene-(CH₂)_(n′)—, or —(CH₂)_(n′)—C(O)—N(R)—(CH₂)_(n′)—; Y is—N(R)-phenylene- or —N(R)—CH₂)_(n′)—; and n′ is 1 to 12. In someembodiments, R is independently hydrogen or C₁₋₆ alkyl.

In some embodiments, a Linker has a reactive functional group which hasa nucleophilic group that is reactive to an electrophilic group presenton M, such as an antibody. Useful electrophilic groups include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a Linker can react with an electrophilic group onan antibody and form a covalent bond to an antibody unit. Usefulnucleophilic groups on a Linker include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a Linker.

Typically, peptide-type Linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press) which is well known in the field ofpeptide chemistry.

Linker intermediates may be assembled with any combination or sequenceof reactions including Spacer, Stretcher, and Amino Acid units. TheSpacer, Stretcher, and Amino Acid units may employ reactive functionalgroups which are electrophilic, nucleophilic, or free radical in nature.Reactive functional groups include, but are not limited to:

where X is a leaving group, e.g. O-mesyl, O-tosyl, —Cl, —Br, —I; ormaleimide.

In some other embodiments, a Linker may be substituted with groups whichmodulate solubility or reactivity. For example, a charged substituentsuch as sulfonate (—SO₃—) or ammonium, may increase water solubility ofthe reagent and facilitate the coupling reaction of the linker reagentwith the antibody or the drug moiety, or facilitate the couplingreaction of Ab-L (antibody-linker intermediate) with D, or D-L(drug-linker intermediate) with Ab, depending on the synthetic routeemployed to prepare the ADC.

In some embodiments, a provided compound of formula II is an ADCprepared with a linker reagent selected from: BMPEO, BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB,SVSB (succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄. Suchreagents are widely described and used, and are commercially availablefrom, for example, Pierce Biotechnology, Inc., Customer ServiceDepartment, P.O. Box 117, Rockford, Ill. 61105 U.S.A., U.S.A.1-800-874-3723, International +815-968-0747. See pages 467-498,2003-2004 Applications Handbook and Catalog. Bis-maleimide reagentsallow the attachment of the thiol group of an antibody, such as acysteine engineered antibody, to a thiol-containing drug moiety, label,or linker intermediate, in a sequential or concurrent fashion. Otherfunctional groups besides maleimide, which are reactive with thiolgroups of an M, L or D unit or intermediate, include iodoacetamide,bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide,isocyanate, and isothiocyanate.

Useful linker reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem.60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; U.S.Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO03/026577; WO 03/043583; and WO 04/032828.

Stretchers of formula L-IIIa can be introduced into a Linker by reactingthe following linker reagents with the N-terminus of an Amino Acid unit:

where n′ is an integer ranging from 1-10 and T is —H or —SO₃Na; or

where n′ is an integer ranging from 0-3;

Among others, Stretcher units can also be introduced into a Linker byreacting the following bifunctional reagents with the N-terminus of anAmino Acid unit:

wherein X is —Br or —I,

Stretcher units of formula can also be introduced into a Linker byreacting the following bifunctional reagents with the N-terminus of anAmino Acid unit:

In some embodiments, an Stretcher unit is introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Isothiocyanate Stretchers of the formula shown below may be preparedfrom isothiocyanatocarboxylic acid chlorides as described in Angew.Chem., (1975) 87(14), 517.

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide Stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative Spacer has the structure:

An exemplary phe-lys(Mtr) dipeptide linker reagent having a maleimideStretcher unit and a p-aminobenzyl self-immolative Spacer unit can beprepared according to Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60, and has the structure:

where Mtr is mono-4-methoxytrityl.

In some embodiments, a linker unit is a heterocycle self-immolativelinker. Exemplary heterocyclic self-immolative linkers are widely known,including those described in U.S. Pat. No. 7,989,434, the entirety ofwhich is hereby incorporated by reference.

In some embodiments, a heterocyclic linker reagents having the structureof formula LL-Ia, LL-IIa, or LLL-IIIa:

wherein:U^(L) is O, S or NR^(6L);Q^(L) is CR^(4L) or N;V^(1L), V^(2L) and V^(3L) are independently CR^(4L) or N provided thatfor

at least one of Q^(L), V¹ and V² is N;T is NH, NR⁶, O or S pending from said drug moiety;R^(1L), R^(2L), R^(3L) and R^(4L) are independently selected from H, F,Cl, Br, I, OH, —N(R^(5L))₂, —N(R^(5L))₃ ⁺, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, —SO₂R^(5L), —S(═O)R^(5L),—SR^(5L), —SO₂N(R^(5L))₂, —C(═O)R^(5L), —CO₂R^(5L), —C(═O)N(R^(5L))₂,—CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈ halosubstituted alkyl,polyethyleneoxy, phosphonate, phosphate, C₁-C₈ alkyl, C₁-C₈ substitutedalkyl, C₂-C₈ alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₁-C₂₀heterocycle, and C₁-C₂₀ substituted heterocycle; or when taken together,R^(2L) and R^(3L) form a carbonyl (═O), or spiro carbocyclic ring of 3to 7 carbon atoms; andR^(5L) and R^(6L) are independently selected from H, C₁-C₈ alkyl, C₁-C₈substituted alkyl, C₂-C₈ alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈alkynyl, C₂-C₈ substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substitutedaryl, C₁-C₂₀ heterocycle, and C₁-C₂₀ substituted heterocycle;where C₁-C₈ substituted alkyl, C₂-C₈ substituted alkenyl, C₂-C₈substituted alkynyl, C₆-C₂₀ substituted aryl, and C₂-C₂₀ substitutedheterocycle are independently substituted with one or more substituentsselected from F, Cl, Br, I, OH, —N(R^(5L))₂, —N(R^(5L))₃ ⁺, C₁-C₈alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, C₁-C₈alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R⁵, —S(═O)R^(5L), —SR^(S),—SO₂N(R^(5L))₂, —C(═O)R^(5L), —CO₂R^(5L), —C(═O)N(R^(5L))₂, —CN, —N₃,—NO₂, C₁-C₈ alkoxy, C₁-C₈ trifluoroalkyl, C₁-C₈ alkyl, C₃-C₁₂carbocyclic, C₆-C₂₀ aryl, C₂-C₂₀ heterocycle, polyethyleneoxy,phosphonate, and phosphate.R^(7L) is the side chain of an amino acid and is optionally protectedwith a protecting group;X^(L) and Y^(L) independently: are H, form a protecting group or form areactive functional group.m^(L) is 1, 2, 3, 4, 5, or 6.

In some embodiments, X^(L) and Y^(L) independently: are H, form aprotecting group selected from Fmoc, Boc, triphenylmethyl, or form areactive functional group selected from N-hydroxysuccinimide,para-nitrophenyl carbonate, para-nitrophenyl carbamate,pentafluorophenyl, haloacetamide, and maleimide.

In some embodiments, a linker unit L comprises a heterocyclicself-immolative moiety selected from formulae LL-I, LL-II, LL-III:

wherein each T^(L) is independently NH, NR^(6L), O or S pending fromsaid drug moiety, and each other variable is independently as definedabove and described herein.

In some embodiments, a linker moiety comprises a heterocyclic“self-immolating moiety” of formula LL-I, LL-II, or LL-III bound to thedrug and incorporates an amide group that upon hydrolysis by anintracellular protease initiates a reaction that ultimately cleaves theself-immolative moiety from the drug such that the drug is released fromthe conjugate in an active form. In some embodiments, a linker moietyfurther comprises a peptide sequence adjacent to the self-immolativemoiety that is a substrate for an intracellular enzyme, for example acathepsin such as cathepsin B, that cleaves the peptide at the amidebond shared with the self-immolative moiety. In some embodiments, a drugmoiety is connected to the self-immolative moiety of the linker via achemically reactive functional group pending from the drug such as aprimary or secondary amine, hydroxyl, sulfhydryl or carboxyl group.

In some embodiments, L has the structure of —Y_(y)—W_(w)—X^(s)—, and aprovided compound of formula II has the structure of formula LL-IV:

M-[Y_(y)—W_(w)—X^(s)-D]_(t)  LL-IV

wherein X^(s) is a heterocyclic self-immolating group having thestructure of LL-I, LL-II, or LL-III.

In some embodiments, M is a cell-specific ligand capable of specificallytargeting a selected cell population, D is a compound of formula I-c orI-d covalently connected to X^(s) and —Y_(y)—W_(w)—X^(s)— is a linkerwherein Y is optionally present as a spacer unit (y is 0 or 1), —W_(w)—is an enzymatically cleavable peptide (amino acid) sequence (w is 1, 2,3, 4, 5 or 6), and X^(s) is a heterocyclic self-immolating groupconnecting the drug moiety D and the enzymatically cleavable peptidesequence —W_(w)—. In some embodiments, the number of drug moieties perligand, i.e. drug loading value t, is 1 to about 8.

Heterocyclic Self-Immolative Moiety (X^(s))

In some embodiments, the drug-ligand conjugates of the invention employa heterocyclic self-immolative moiety (X^(s)) covalently linked to thedrug moiety and the cleavable peptide sequence moiety. In someembodiments, a self-immolative moiety is a bifunctional chemical groupwhich is capable of covalently linking together two spaced chemicalmoieties into a normally stable molecule, releasing one of said spacedchemical moieties from the molecule by means of enzymatic cleavage; andfollowing said enzymatic cleavage, spontaneously cleaving from theremainder of the bifunctional chemical group to release the other ofsaid spaced chemical moieties. In some embodiments, a self-immolativemoiety is covalently linked at one of its ends, directly or indirectlythrough a Spacer unit, to the ligand by an amide bond and covalentlylinked at its other end to a chemical reactive site (functional group)pending from the drug. In some embodiments, the derivatization of thedrug with the self-immolative moiety may render the drug lesspharmacologically active (e.g. less toxic) or not active at all untilthe drug is cleaved.

In some embodiments, the conjugate is stable extracellularly, or in theabsence of an enzyme capable of cleaving the amide bond of theself-immolative moiety. Upon entry into a cell, or exposure to asuitable enzyme, the amide bond is cleaved initiating a spontaneousself-immolative reaction resulting in the cleavage of the bondcovalently linking the self-immolative moiety to the drug, to therebyeffect release of the drug in its underivatized or pharmacologicallyactive form. In one embodiment, the self-immolative linker is coupled tothe ligand, through an enzymatically cleavable peptide sequence thatprovides a substrate for an intracellular enzyme to cleave the amidebond to initiate the self-immolative reaction.

In some embodiments, a self-immolative moiety in a provided compoundeither incorporate one or more heteroatoms and thereby provides improvedsolubility, improves the rate of cleavage and decreases propensity foraggregation of the conjugate.

In some embodiments, when T^(L) is NH, it is derived from a primaryamine (—NH₂) pending from the drug moiety (prior to coupling to theself-immolative moiety). In some embodiments, when T^(L) is N, it isderived from a secondary amine (—NH—) from the drug moiety (prior tocoupling to the self-immolative moiety). In some embodiments, when T^(L)is O or S, it is derived from a hydroxyl (—OH) or sulfhydryl (—SH) grouprespectively pending from the drug moiety prior to coupling to theself-immolative moiety.

Not to be limited by theory or a particular mechanism, the presence ofelectron-withdrawing groups on the heterocyclic ring of formula LL-I,LL-II or LL-III linkers may moderate the rate of cleavage.

In some embodiments, a self-immolative moiety is the group of formulaLL-I in which Q^(L) is N, and U^(L) is O or S. In some embodiments, suchgroup has a non-linearity structural feature which improves solubilityof the conjugates. In this context, in some embodiments, R^(1L) may beH, methyl, nitro, or CF₃ while T^(L) is N or NH pending from the drugmoiety D. In one embodiment, Q^(L) is N and U^(L) is O thereby formingan oxazole ring and R^(1L) is H. In another embodiment, Q^(L) is N andU^(L) is S thereby forming a thiazole ring optionally substituted atR^(1L) with an Me or CF₃ group and T^(L) is N or NH pending from drugmoiety D. It will be understood that when T^(L) is NH, it is derivedfrom a primary amine (—NH₂) pending from the drug moiety (prior tocoupling to the self-immolative moiety) and when T^(L) is N, it isderived from a secondary amine (—NH—) from the drug moiety (prior tocoupling to the self-immolative moiety). Similarly, when T^(L) is O orS, it is derived from a hydroxyl (—OH) or sulfhydryl (—SH) grouprespectively pending from the drug moiety prior to coupling to theself-immolative moiety.

In another exemplary embodiment, the self-immolative moiety is the groupof formula LL-II in which Q^(L) is N and V^(1L) and V^(2L) areindependently N or CH and T^(L) is N or NH. In another embodiment,Q^(L), V^(1L) and V^(2L) are each N. In another embodiment, Q^(L) andV^(1L) are N while V^(2L) is CH. In another embodiment, Q^(L) and V^(2L)are N while V^(1L) is CH.

In another embodiment, Q and V^(1L) are both CH and V^(2L) is N. Inanother embodiment, Q^(L) is N while V^(1L) and V^(2L) are both CH.

In another embodiment, the self-immolative moiety is the group offormula LL-III in which Q^(L), V^(1L), V^(2L), and V^(3L) are eachindependently N or CH and T^(L) is N or NH. In another embodiment Q^(L)is N while V^(1L), V^(2L) and V^(3L) are each N. In another embodiment,Q^(L), V^(1L), and V^(2L) are each CH while V^(3L) is N. In anotherembodiment Q^(L), V^(2L) and V^(3L) are each CH while V^(1L) is N. Inanother embodiment, Q^(L), V^(1L) and V^(3L) are each CH while V^(2L) isN. In another embodiment, Q^(L) and V^(2L) are both N while V^(1L) andV^(3L) are both CH. In another embodiment Q^(L) and V^(2L) are both CHwhile V^(1L) and V^(3L) are both N. In another embodiment, Q^(L) andV^(3L) are both N while V^(1L) and V^(2L) are both CH.

Cleavable Peptide Sequence (Z_(M))

In the conjugate of Formula IV, each m is independently 1, 2, 3, 4, 5 or6. In exemplary embodiments, m may be 1, 2 or 3, to form single aminoacid, dipeptide, and tripeptide amino acid units, respectively Aminoacid units Z are selected from natural and non-natural amino acids. Theside chain-bearing carbon may be in either D or L (R or S) configurationAmino acid unit Z may be alanine, 2-amino-2-cyclohexylacetic acid,2-amino-2-phenylacetic acid, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, γ-aminobutyric acid, α,α-dimethylγ-aminobutyric acid, β,β-dimethyl γ-aminobutyric acid, ornithine, andcitrulline (Cit) Amino acid unit Z optionally includes protected formsof amino acids where reactive functionality of the side chains areprotected. Protected amino acid reagents and intermediates are wellknown, including lysine-protected with acetyl, formyl, triphenylmethyl(trityl), and monomethoxytrityl (MMT). Other protected amino acid unitsinclude arginine-protected tosyl or nitro group, ornithine-protectedwith acetyl or formyl groups.

Each Z_(m) unit independently has the formula denoted below in thesquare brackets, where m is an integer ranging from 0 to 6:

In some embodiments, —W_(w)— is a cleavable peptide sequence.

In some embodiments, in the conjugate of formula LL-IV, each w isindependently 1, 2, 3, 4, 5 or 6. In exemplary embodiments, m may be 1,2 or 3, to form single amino acid, dipeptide, and tripeptide amino acidunits, respectively. Amino acid units W are selected from natural andnon-natural amino acids. A side chain-bearing carbon may be in either Dor L (R or S) configuration. An amino acid unit W may be alanine,2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, γ-aminobutyricacid, α,α-dimethyl γ-aminobutyric acid, β,β-dimethyl γ-aminobutyricacid, ornithine, and citrulline (Cit). Suitable amino acid unit Woptionally includes protected forms of amino acids where reactivefunctionality of the side chains are protected. Protected amino acidreagents and intermediates are well known, including lysine-protectedwith acetyl, formyl, triphenylmethyl (trityl), and monomethoxytrityl(MMT). Other protected amino acid units include arginine-protected withtosyl or nitro group, ornithine-protected with acetyl or formyl groups,etc.

In some embodiments, each —W-unit independently has the structure of:

In some embodiments, w is 0-6.

The peptide unit sequence —W_(w)— is specifically tailored so that itwill be selectively enzymatically cleaved from the drug moiety by one ormore of the cellular proteases. The amino acid residue chain length ofthe peptide linker ranges from that of a single amino acid to abouteight amino acid residues. Exemplary enzymatically-cleavable peptidesequences include Gly-Gly, Phe-Lys, Val-Lys, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit,Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly-Val-Cit,Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N 9-tosyl-Arg, and Phe-N9-Nitro-Arg, in either orientation. Numerous specific cleavable peptidesequences suitable for use can be designed and optimized in theirselectivity for enzymatic cleavage by a particular intracellular enzymee.g. a tumor-associated protease. Cleavable peptides for use alsoinclude those which are optimized toward the proteases, cathepsin B, Cand D, such as Phe-Lys, Ala-Phe, and Val-Cit. In some embodiments, apeptide sequence for use is tripeptide D-Ala-Phe-Lys, which isselectively recognized by the tumor-associated protease plasmin, whichmay be involved in tumor invasion and metastasis (de Groot, et al (2002)Molecular Cancer Therapeutics 1(11):901-911; de Groot, et al (1999) J.Med. Chem. 42(25):5277-5283).

In some embodiments, conjugates of formula LL-IV optionally incorporatea spacer unit Y (i.e. y is not 0). In some embodiments, Y is a divalentmoiety that couples the N-terminus of the cleavable peptide (—W_(w)—) tothe ligand M. In some embodiments, the spacer unit is of a length thatenables the cleavable peptide sequence to be contacted by the cleavingenzyme (e.g. cathepsin B) and the hydrolysis of the amide bond couplingthe cleavable peptide to the self-immolative moiety X^(s). In someembodiments, a Spacer unit Y is covalently bound to W_(w) via an amidebond. In some embodiments, a spacer unit is a bond and ligand M isdirectly and covalently attached to the self-immolative moiety X^(s). Inthis case, the ligand M and the self-immolative moiety X^(s) form anamide bond that upon proteolytic cleavage initiates the self-immolativereaction and the ultimate release of the drug D.

In some embodiments, a spacer unit Y is covalently bound to a functionalgroup pending from the ligand M such as an amine (e.g. —NH₂ from a Lysresidue), a carboxyl (—COOH from an Asp or Glu residue) or a sulfhydryl(e.g. —SH from a Cys residue) which forms an amide or a thioether ordisulfide group. Spacer units may comprise a divalent radical such asalkyldiyl, aryldiyl, heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide.

Conjugates of the invention in which the spacer unit Y is reacted with asulfhydryl functional group of ligand M (for example when M is Cyscontaining peptide or a reduced antibody) to form a thioether linkageinclude those represented by formulae LL-Va-Ve), in which spacer unit Yis the compound in brackets.

In some embodiments, Y has the structure of

In some embodiments, a provided compound of formula II has the structureof formula LL-Va:

In some embodiments, Y is maleimidocaproyl (MC) (where R¹⁷ is —(CH₂)₅—;e.g., made from maleimidocaproyl-N-hydroxysuccinimide (MC-NHS)):

In some embodiments, Y is maleimido-propanoyl (MP):

In some embodiments, R¹⁷ is —(CH₂CH₂O)_(r)—CH₂— and r is 2, and Y is

In some embodiments, R¹⁷ is —(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— whereR^(b) is H and each r is 2, and Y is

In some embodiments, Y is SMCC (for example, made from succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate):

In some embodiments, a compound of formula II has the structure of

In some embodiments, Y has the structure of

(In some embodiments, made from m-maleimidobenzoyl-N-hydroxysuccinimideeste (MBS)). In some embodiments, a compound of formula II has thestructure of

In some embodiments, Y has the structure of

(In some embodiments, made from succinimidyl4-(p-maleimidophenyl)butyrate (SMPB)). In some embodiments, a compoundof formula II has the structure of

In some embodiments, Y has the structure of

(In some embodiments, made from made fromN-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB)). In some embodiments,a compound of formula II has the structure of

In some other embodiments, spacer unit Y and ligand M are linked via athioether group. In some embodiments, such a compound may be prepared byreacting a sulfhydryl functional group pending from ligand M with anactivated disulfide-containing precursor of spacer unit Y. Exemplaryconjugates of this type include

In some embodiments, Y has the structure of

(In some embodiments, made from made from4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT)).In some embodiments, a compound of formula II has the structure of

In some embodiments, Y has the structure of

(In some embodiments, made from succinimidyl6-[3-(2-pyridyldithio)-propionamide]hexanoate (LC-SPDP)). In someembodiments, a compound of formula II has the structure of

In some embodiments, Y has the structure of

In some embodiments, a compound of formula II has the structure of

In some embodiments, Y has the structure of

In some embodiments, Y, and/or a provided conjugate, is prepared with across-linking reagent selected from BMPEO, BMPS, EMCS, GMBS, HBVS,LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB,SVSB (succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimide reagentsDTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄. Many cross-linkingreagents are commercially available, for example, from PierceBiotechnology, Inc., Customer Service Department, P.O. Box 117,Rockford, Ill. 61105 USA, 1-800-874-3723, International +815-968-0747.See pages 467-498, 2003-2004 of the Applications Handbook and Catalog.In some embodiments, bis-maleimide reagents allow the attachment of athiol group of a cysteine residue of an M unit, such as an antibody, toa thiol-containing drug moiety or linker intermediate, in a sequentialor concurrent fashion. Other functional groups besides maleimide, whichare reactive with a thiol group of M (e.g., an antibody), drug moiety,or linker intermediate include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

Useful spacer reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; U.S. Pat. No. 6,214,345 to Firestone et al;WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583;and WO 04/032828.

A provided compound in which the spacer unit Y is coupled to ligand Mvia an amide group may be prepared by reacting a free amine functionalgroup on ligand M with an active ester containing precursor of spacerunit Y. For example, a carboxyl group on spacer unit may be activated byreacting with N-hydroxysuccinimide and then reacted with M-NH₂ to form aconjugate in which M and Y or coupled by way of an amide group.

Useful functional groups on an antibody for linking to the spacer unit,either naturally or via chemical manipulation include, but are notlimited to, sulfhydryl (—SH), amino, hydroxyl, the anomeric hydroxylgroup of a carbohydrate, and carboxyl. In some embodiments, the reactivefunctional groups on the antibody are sulfhydryl and amino. Sulfhydrylgroups can be generated by reduction of an intramolecular cysteinedisulfide bond of an antibody, or can be generated by reaction of anamino group of a lysine moiety of an antibody using 2-iminothiolane(Traut's reagent) or another sulfhydryl generating reagent. In someembodiments, a sulfhydryl group is generated by modifying an antibody'samino acid sequence, for example, by replacing an amino acid residuewith a cysteine residue. In some embodiments, an antibody is a cysteineengineered antibody.

In some embodiments, the Spacer unit is linked to the antibody unit viaa disulfide bond between a sulfur atom of the Antibody unit and a sulfuratom of the Spacer unit, for example, as in

In some embodiments, t, the average number of drug moieties per antibodyunit is from 1 to about 8.

In some other embodiments, the reactive group of the Spacer contains areactive site that can form a bond with a primary or secondary aminogroup of an antibody. Examples of these reactive sites include, but arenot limited to, activated esters such as succinimide esters,4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenylesters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. Exemplary such Spacer units are depicted in ADCs havingthe structure of AbC(O)NH—R¹⁷—C(O)—W_(w)—X^(s)-D]_(t) andAbC(S)NH—R¹⁷—C(O)—W_(w)—X^(s)-D]_(t).

In some embodiments, a reactive group of the Spacer reacts with anelectrophilic group, such as an aldehyde, ketone, acetal, or ketal groupof an antibody, or a sugar (carbohydrate) of a glycosylated antibody. Insome embodiments, a carbohydrate can be mildly oxidized using a reagentsuch as sodium periodate and the resulting (—CHO) unit of the oxidizedcarbohydrate can be condensed with a Spacer that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko, T. et al (1991)Bioconjugate Chem 2:133-41. In some embodiments, an electrophilic group,such as an aldehyde or ketone group, is introduced through incorporationof an unnatural amino acid. For example, US Patent ApplicationPublication US20100210543 and US20120183566 describes proteins,including antibodies, bearing at least one aldehyde groups, andpreparation methods and processes thereof. Exemplary such Spacer unitsare depicted in compounds having the structure of

In some embodiments, L comprises an —S—S— moeity.

In some embodiments, a linker unit (L) comprises one of the followingmoieties (Singh et al, Recent Trends in Targeted Anticancer Prodrug andConjugate Design, Curr Med Chem. 2008; 15(18): 1802-1826):

In some embodiments, L comprises a group of

Exemplary M embodiments (ligand, e.g., antibodies) and/or L embodiments(linker units, e.g., moieties linking a drug or an active unit to aligand, for example, an antibody) also include those described in U.S.Pat. Nos. 5,475,092, 6,214,345, 7,659,241, 7,223,837, 7,705,045,8,158,590, 8,012,978, 8,337,856, 7,750,116, 7,659,241, 7,989,434,7,851,437, 7,829,531, 7,754,681, 7,714,016, 7,585,834, 7,553,816,7,091,186, 6,855,689, 6,759,509, 6,677,435, 6,268,488, 5,877,158,7,375,078, 7,547,768, 7,754,441, 7,803,915, 7,749,504, 7,662,936,7,855,275, 7,521,541, 7,479,544, 7,723,485, 7,989,595, 7,304,032,6,897,034, 7,858,759, 7,842,789, 7,507,405, 7,214,776, 7,122,636,7,834,154, 7,723,485, 7,183,076, 7,018,809, 6,582,928, 5,929,211,7,087,840, 7,964,567, 7,964,566, 7,745,394, 7,256,257, 7,427,399,7,494,646, 7,541,442, 7,595,379, 7,968,090, 7,811,565, 6,103,236,7,696,313, 7,214,663, 7,115,573, 7,691,962, 7,893,023, 7,816,317,7,319,139, and 6,870,033; US Patent Application Publications US2011/0137017, US 2011/0195021, US 2011/0195022, US 2008/0050310, US2009/0068202, US 2009/0280056, US 2010/0215669, US 2011/0142859, US2011/0135667, and US 2008/0267981; and International Patent ApplicationPublications WO 2007/089149, WO 2009/017394, WO 2010/062171, WO2011/133039, WO 2012/177837, WO 2012/149412, WO 2012/145112, WO2012/138749, WO 2012/135675, WO 2012/138537, WO 2012/135740, WO2012/135517, WO 2012/135522, WO 2012/128868, WO 2012/112687, WO2012/112708, WO 2012/078868, WO 2012/061590, WO 2012/058592, WO2012/019024, WO 2011/162933, WO 2011/112978, WO 2011/106528, WO2011/100403, WO 2011/100398, WO 2011/091286, WO 2011/050180, WO2010/126551. WO 2010/141566, WO 2010/128087, WO 2010/126552, WO2009/134870, WO 2010/008726, WO 2009/134952, WO 2009/134976, WO2009/134977, WO 2009/080830, WO 2009/080832, WO 2009/080831, WO2007/024536, WO 2006/086733, WO 2007/024222, WO 2007/019232, WO2005/037992, WO 2004/110498, WO 2005/009369, WO 2004/043344, WO2004/016801, WO 2004/013093, WO 2004/005470, WO 2003/106621, WO2003/068144, WO 2002/098883, WO 2001/024763, WO 2013055993, WO2013055990, WO 2013049410, WO 2012/078688, WO 2012/054748, WO2012/047724, WO 2011/130613, WO 2011/038159, WO 2010/111018, WO2010/081004, WO 2009/135181, WO 2009/117531, WO 2009/052431, WO2009/048967, WO 2008/070593, WO 2008/025020, WO 2007/137170, WO2007/103288, WO 2007/075326, WO 2007/062138, WO 2007/030642, WO2007/011968, WO 2007/008848, WO 2007/008603, WO 2006/128103, WO2006/113909, WO 2006/065533, WO 2006/132670, WO 2006/044643, WO2005/084390, WO 2005/082023, WO 2005/077090, WO 2005/070026, WO2005/081711, WO 2005/001038, WO 2004/010957, WO 2002/043661, WO2004/090113, WO 2004/050867, and WO 2004/032828. In some embodiments, Mis an antibody described in one of the above-referenced patents and/orpatent applications. In some embodiments, L is a moiety described in oneof the above-referenced patents and/or patent applications that links aligand (e.g., an antibody) to a drug unit (or other active unit, such asa cytotoxic unit). In some embodiments, L is a moiety described in oneof the above-referenced patents and/or patent applications that links anantibody to a drug unit.

In some embodiments, L is a polymer unit. In some embodiments, L is apolyalkylene glycol linker. In some embodiments, L is a PEG linker. Insome embodiments, L is a monodispersed PEG linker. Exemplary polymericmoieties, including PEG moieties, that can be used as linker unitsinclude those described in U.S. Pat. Nos. 7,119,162, 7,030,082,6,858,580, 6,835,802, 6,815,530, 7,888,536, 6,620,976, 7,846,893, and USPatent Application Publications US 20110124844, US 20110118480, and US20090203584.

In some embodiments, the present invention recognizes the challenges forpreparing ETP or thiodiketopiperazine alkaloids or derivatives oranalogs thereof. In some embodiments, the present invention provides amethod for preparing ETP or thiodiketopiperazine alkaloids orderivatives or analogs thereof. In some embodiments, the presentinvention provides a method for preparing a provide compound. In someembodiments, the present invention provides new reagents for preparingETP or thiodiketopiperazine alkaloids or derivatives or analogs thereof.In some embodiments, the present invention provides new reagents forpreparing a provided compound. In some embodiments, a provided methodand/or reagent provides unexpectedly high synthetic efficiency, forexample, in terms of product yield and/or purity.

In some embodiments, the present invention provides methods for flexibleand scalable synthesis of ETP or thiodiketopiperazine alkaloids orderivatives or analogs thereof, for example, a provided compound offormula I-a, I-b, I-c, I-d, II, or III. In some embodiments, the presentinvention provides a method for scalable synthesis, e.g., >5 g, >6 g, >7g, >8 g, >9 g, >10 g, >11 g, >12 g, >13 g, >14 g, >15 g, >16 g, >17g, >18 g, >19 g, or >20 g, >15 g or >20 g scale, of anerythro-β-hydroxytryptophan compound, an intermediate useful for thepreparation of ETP or thiodiketopiperazine compounds, or derivatives oranalogs thereof. In some embodiments, the present invention provides amethod for scalable synthesis of an erythro-β-hydroxytryptophancompound, comprising step S-I-1:

wherein each variable is independently as defined above and describedherein. In some embodiments, INT-Ib is enantiomerically pure. In someembodiments, INT-1b is racemic. In some embodiments, INT-1b containsmore of one enantiomer than the other. In some embodiments, thestereochemistry of the

moiety remains essentially the same during step S-I-1. It is understoodby a person of ordinary skill in the art that among other methods,enantiomeric purity of INT-1b can be controlled by selectively using oneof the two enantiomers, or adjusting the relative amounts of the twoenantiomers of INT-Ic.

In some embodiments, step S-I-1 comprises the use of a metal salt. Insome embodiments, step S-I-1 is mediated by a titanium salt. In someembodiments, a titanium salt is TiCl(OEt)₃. In some embodiments, stepS-I-1 is conducted at a temperature below room temperature. In someembodiments, step S-I-1 is conducted at about 0° C. In some embodiments,step S-I-1 comprises the use of a base. In some embodiments, a base isNEt₃. An exemplary condition is TiCl(OEt)₃, NEt₃, CH₂Cl₂, 0° C.

In some embodiments, a provided method for scalable synthesis of anerythro-β-hydroxytryptophan compound further comprises step S-I-2, whichcomprises removing the auxiliary group

In some embodiments, the auxiliary group is recovered, e.g., as

and re-used. In some embodiments, the auxiliary group is removed underacid conditions. In some embodiments, an acid is HCl. An exemplaryacidic condition for step S-I-2 is 2 N HCl, THF.

In some embodiments, step S-I-2 further comprises protection of thehydroxyl group in formula INT-Ib. In some embodiments, a protectiongroup is —OSi(R)₃. In some embodiments, a protection group is -TBS. Anexemplary condition for introducing the protection group is: TBSOTf,2,6-lutide, CH₂Cl₂, 23° C. In some embodiments, step S-I-2 provides acompound having the structure of formula INT-Ia:

wherein R^(6′) is —OR or —OSi(R)₃, and each other variable isindependently as defined above and described herein. In someembodiments, INT-Ia is enantiomerically pure. In some embodiments,INT-Ia has an ee value greater than 0. In some embodiments, INT-Ia isracemic.

In some embodiments, an erythro-β-hydroxytryptophan compound is preparedaccording to scheme I set forth below:

In some embodiments, each of INT-Ia, INT-1b and INT-1c isenantiomerically pure. It is understood by a person of ordinary skill inthe art that in Scheme I, INT-Ic can be either of its enantiomers, andthe configurations of the chiral centers in INT-Ic can remainessentially the same during the transformation.

In some embodiments, n is 0. In some embodiments, R^(3′) is R³. In someembodiments, R³ is —SO₂Ph. In some embodiments, R is ethyl. In someembodiments, R^(6″) is —OH. In some embodiments, R^(6″) is -OTBS.

In some embodiments, one of R⁶ and R^(6′) is —OR or —OSi(R)₃. Withoutwishing to be limited by theory, Applicants note that in someembodiments such compounds cannot be prepared efficiently usingintermolecular Friedel-Crafts indolylation at the C3 position possiblydue to, for example, the inductive and steric effects of theC-12-hydroxyl group:

In some embodiments, the present invention provides methods forpreparation of a compound having the structure of formula I-c, whereinone of R⁶ and R^(6′) is —OR or —OSi(R)₃. In some embodiments, a compoundof I-c is a compound of I-a. In some embodiments, R⁴ is an optionallysubstituted group selected from phenyl, an 8-14 membered bicyclic orpolycyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments,

is a single bond.

In some embodiments, the present invention provides a method forpreparing a compound of formula I-e:

comprising steps of:

wherein:

-   R^(4′) is an optionally substituted group selected from phenyl, an    8-14 membered bicyclic or polycyclic aryl ring, a 5-6 membered    monocyclic heteroaryl ring having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-14 membered    bicyclic or polycyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R^(4I) is an optionally substituted bivalent group selected from    phenylene, an 8-14 membered bicyclic or polycyclic arylene ring, a    5-6 membered monocyclic heteroarylene ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or an 8-14    membered bicyclic or polycyclic heteroarylene ring having 1-5    heteroatoms independently selected from nitrogen, oxygen, or sulfur;-   R^(4L) is a leaving group;-   R^(4P) is an optionally substituted group selected from phenyl, an    8-14 membered bicyclic or polycyclic aryl ring, a 5-6 membered    monocyclic heteroaryl ring having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or an 8-14 membered    bicyclic or polycyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R^(6″) is —OR or —OSi(R)₃;-   R^(6L) is —Si(R)₂—O—;-   R^(8′) is hydrogen or R⁸;-   R^(9′) is hydrogen or R⁹; and-   each other variable is independently as defined above and described    herein;

In some embodiments, each of R^(4′), R^(4P) and R^(4I) is electron-rich.In some embodiments, each of R^(4′) and R^(4P) is independently anoptionally substituted indolyl group. In some embodiments, R^(4′) isoptionally substituted 3-indolyl. In some embodiments, R^(4′) issubstituted 3-indolyl. In some embodiments, R^(4′) is substituted3-indolyl comprising N-substitution. In some embodiments, R^(4P) isoptionally substituted 2-indolyl. In some embodiments, R^(4P) issubstituted 2-indolyl. In some embodiments, R^(4P) is substituted2-indolyl comprising N-substitution. In some embodiments, R^(4I) is anoptionally substituted bivalent indole ring moiety. In some embodiments,R^(4I) is an optionally substituted bivalent 2,3-indole ring moiety. Insome embodiments, R^(8′) is hydrogen. In some embodiments, R^(9′) ishydrogen. In some embodiments, R^(6L) is —Si(Me)₂-O—. In someembodiments, R^(6L) is —Si(Et)₂-O—.

Exemplary leaving group are widely known and used in organic synthesis.In some embodiments, R^(4L) is halogen. In some embodiments, R^(4L) is—Cl. In some embodiments, R^(4L) is —Br. In some embodiments, R^(4L) is—I. In some embodiments, R^(4L) is —OS(O)₂R.

In some embodiments, a compound of formula I-e has the structure offormula I-e-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I-f has the structure offormula I-f-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, a compound of formula I-g has the structure offormula I-g-1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as described in classes and subclasses herein, both singlyand in combination.

In some embodiments, step S-II-1 comprises an intramolecularFriedel-Crafts reaction. In some embodiments, an intramolecularFriedel-Crafts reaction is mediated by a silver salt. In someembodiments, a silver salt is AgBF₄. In some embodiments, anintramolecular Friedel-Crafts reaction is conducted at a temperaturebelow room temperature. In some embodiments, an intramolecularFriedel-Crafts reaction is conducted at about 0° C. In some embodiments,step S-II-1 comprises the use of a base. In some embodiments, a base is2,6-di-tert-butyl-4-methylpyridine (DTBMP). An exemplary condition isAgBF₄, DTBMP, EtNO₂, 0° C.

In some embodiments, step S-II-2 comprises breaking one or more bonds ofR^(6L). In some embodiments, step S-II-2 comprises the remove of R^(6L),i.e, R^(6L) is replaced with two —H. In some embodiments, a compound ofI-f is converted to a compound of I-e under acidic conditions. In someembodiments, a compound of I-f is converted to a compound of I-e at atemperature higher than room temperature. In some embodiments, acompound of I-f is converted to a compound of I-e under acidicconditions and at a temperature higher than room temperature. Anexemplary condition is 6 N HCl, THF, 80° C.

In some embodiments, step S-II-2 is carried out subsequently to stepS-II-1. In some embodiments, step S-II-2 is conducted concurrently withstep S-II-1. In some embodiments, steps S-II-1 and S-II-2 are carriedout in one pot.

In some embodiments, the present invention provides a method forpreparing triketopiperazine. In some embodiments, a provided methodcomprises use of a permanganate reagent. In some embodiments, apermanganate reagent is bis(pyridine)silver(I) permanganate.

In some embodiments, the present invention provides a method forpreparing a triketopiperazine having the structure of formula I-h:

-   or a pharmaceutically acceptable salt thereof, wherein each variable    is independently as described in classes and subclasses herein, both    singly and in combination,    comprising providing a permanganate reagent.

In some embodiments, the present invention provides a method forpreparing a triketopiperazine having the structure of formula I-h,further comprising providing a compound having the structure of I-i:

wherein each variable is independently as described in classes andsubclasses herein, both singly and in combination.

In some embodiments, in a provided method R⁶ is —H and R^(6′) is —OH or—OR. In some embodiments, in a provided method R⁴ is optionallysubstituted, N-substituted indolyl, R⁶ is —H and R^(6′) is —OH or —OR.In some embodiments, an N-substituent is an electron-withdrawing group.In some embodiments, in a provided method R⁶ is —H, R^(6′) is —OH or—OR, and the newly installed —OH in formula I-h is trans to R^(6′). Insome embodiments, in a provided method R⁶ is —H, R^(6′) is —OH or —OR,and the newly installed —OH in formula I-h has inverted stereochemistryof the originating C—H stereochemistry (hydroxylation with inversion ofthe originating C—H stereochemistry). An exemplary transformation isillustrated below:

In some embodiments, the present invention provides new reagents andmethods for sulfidation. In some embodiments, a provided reagent is aproduct of the addition of thiols to enones. In some embodiments, aprovided reagent has the structure of HSC(R)₂C(R)₂C(O)R. In someembodiments, a provided reagent has the structure of HSCH₂CH₂C(O)R. Insome embodiments, a provided reagent is HSCH₂CH₂C(O)Me. In someembodiments, a provided reagent is HSCH₂CH₂C(O)Ph.

In some embodiments, the present invention provides a method comprisingsteps of:

-   -   (i) providing a first compound comprising at least one hydroxyl        group or protected hydroxyl group;    -   (ii) providing a second compound having the structure of        HSC(R)₂C(R)₂C(O)R;    -   (iii) replacing at least one hydroxyl group or protected        hydroxyl group or derivatives thereof in the first compound with        —SC(R)₂C(R)₂C(O)R of the second compound to provide a third        compound; and    -   (iv) optionally removing the —C(R)₂C(R)₂C(O)R group from the        third compound.

In some embodiments, a provided method is a method for sulfidation. Insome embodiments, a first compound comprises a diketopiperazine moiety.In some embodiments, a first compound comprises a diketopiperazinemoiety and two hydroxyl groups, wherein each of the hydroxyl groups isindependently bonded to an α-carbon of an carbonyl group of thediketopiperazine moiety. In some embodiments, a first compoundcomprising a moiety having the structure of

In some embodiments, a first compound comprising a moiety having thestructure of

In some embodiments, a first compound comprising a moiety having thestructure of

wherein R^(PG) is a protecting group for hydroxyl. In some embodiments,a first compound comprising a moiety having the structure of

In some embodiments, a first compound has the structure of

In some embodiments, a first compound has the structure of

wherein R is Boc and R′ is Piv. In some embodiments, a second compoundhas the structure of HSCH₂CH₂C(O)R. In some embodiments, a secondcompound is HSCH₂CH₂C(O)Me. In some embodiments, a second compound isHSCH₂CH₂C(O)Ph. In some embodiments, a third compound comprises a moietyhaving the structure of

In some embodiments, a third compound has the structure of

In some embodiments, a third compound is

In some embodiments, step (iii) comprises use of nitromethane as asolvent. In some embodiments, step (iii) is stereoselective. In someembodiments, step (iii) is diastereoselective. In some embodiments, step(iv) comprises the use of an amine. In some embodiments, an amine hasthe structure of HN(R)₂. In some embodiments, an amine has the structureof HN(R)₂, wherein the two R groups are taken together to form anoptionally substituted 5-6 membered saturated ring. In some embodiments,an amine is pyrrolidine. In some embodiments, step (iv) comprises ap-elimination reaction facilitated by enamine catalysis. In someembodiments, step (iv) comprises use of free thiol. In some embodiments,step (iv) is conducted under a mild condition that does not disrupt anN-protecting group of an indolyl moiety. In some embodiments, step (iv)is conducted under a mild condition that does not disrupt anN-protecting group of an indolyl moiety, wherein the protecting group is—S(O)₂R. In some embodiments, step (iv) is conducted under a mildcondition that does not disrupt an N-protecting group of an indolylmoiety, wherein the protecting group is —S(O)₂Ph.

In some embodiments, a provided compound of formula I-a, I-b, I-c or I-dis a compound selected from Table 1, below, or a pharmaceuticallyacceptable salt thereof.

TABLE 1 Exemplary compounds.

In some embodiments, a provided of formula I-a, I-b, I-c or I-d is acompound selected from Table 2, below, or a pharmaceutically acceptablesalt thereof:

TABLE 2 Exemplary compounds.

In some embodiments, a provided compound of formula I-c or I-d or D hasthe structure of those illustrated in Table 3:

TABLE 3 Exemplary structures.

R = H, R′ = OH, n = 2 Verticillin A (2) R = H, R′ = H, n = 212,12′-Dideoxyverticillin A (3) R = OH, R′ = H, n = 2 Chaetocin A (4) R= OH, R′ = H, n = 3 Chaetocin C (5) R = OH, R′ = H, n = 412,12′-Dideoxychetracin A (6)

R = H, R′ = OH, n = 2 Bionectin A (9) R = H, R′ = H, n = 212,-Deoxybionectin A (10) R = i-Pr, R′ = OH, n = 3 Leptosin E (11) R =Me, R′ = H, n = 2 Glioclatine (12) R = Me, R′ = OH, n = 4 Gliocladine E(13)

14, n = 2, R = H, R¹ = SO₂Ph 3, n = 2, R = H, R¹ = H 4, n = 2, R = OH,R¹ = H 15, n = 2, R = OAc, R¹ = H 5, n = 3, R = OH, R¹ = H 16, n = 3, R= OAc, R¹ = H 17, n = 3, R = OAc, R¹ = COCF₃ 6, n = 4, R = OH, R¹ = H

24, n = 2, R = SO₂Ph, R¹ = Boc 25, n = 1, R = SO₂Ph, R¹ = H 26, n = 2, R= SO₂Ph, R¹ = H 27, n = 3, R = SO₂Ph, R¹ = H 28, n = 4, R = SO₂Ph, R¹ =H 10, n = 2, R = H, R¹ = H 29, n = 3, R = H, R¹ = H

30, R = Br 31, R = F 32, R = pyrrol-3′-yl 33, R = p-MeOPh

34, R = Br 35, R = F

36, X = S 37, X = O 38, X = H,H

7, R = H, R¹ = Me, R² = H 39, R = SO₂Ph, R¹ = Me, R² = H 40, R = SO₂Ph,R¹ = MOM, R² = H 41, R = H, R¹ = MOM, R² = H 42, R = SO₂Ph, R¹ = MEM, R²= Boc 43, R = SO₂Ph, R¹ = Bn, R² = Boc 44, R = SO₂Ph, R¹ = Ac, R² = H45, R = SO₂Ph, R¹ = SMe, R² = SMe 46, R = SO₂Ph, R¹ = SMe, R² = H

44, R = SO₂Ph, R¹ = Ac, R² = H 45, R = SO₂Ph, R¹ = SMe, R² = SMe 46, R =SO₂Ph, R¹ = SMe, R² = H

60, R = indol-3′-yl 61, R = n-Pr

62, R = indol-3′-yl 63, R = n-Pr

64, R = indol-3′-yl 65, R = n-Pr

66, R = indol-3′-yl 67, R = n-Pr

In some embodiments, the present invention provides an antibody-drugconjugate, wherein the drug unit is selected from a structure in Table1, 2 or 3. In some embodiments, the present invention provides anantibody-drug conjugate, wherein the drug unit is selected from astructure in Table 1. In some embodiments, the present inventionprovides an antibody-drug conjugate, wherein the drug unit is selectedfrom a structure in Table 2. In some embodiments, the present inventionprovides an antibody-drug conjugate, wherein the drug unit is selectedfrom a structure in Table 3. In some embodiments, the present inventionprovides an antibody-drug conjugate, wherein the drug unit has thestructure of formula I-a or I-b. In some embodiments, the drug unit hasthe structure of formula I-a-1. In some embodiments, the drug unit hasthe structure of formula I-a-2. In some embodiments, the drug unit hasthe structure of formula I-a-3. In some embodiments, the drug unit hasthe structure of formula I-a-4. In some embodiments, the drug unit hasthe structure of formula I-a-5. In some embodiments, the drug unit hasthe structure of formula I-b-1. In some embodiments, the drug unit hasthe structure of formula I-b-2. In some embodiments, the drug unit hasthe structure of formula I-b-3. In some embodiments, the drug unit hasthe structure of formula I-b-4. In some embodiments, the drug unit hasthe structure of formula I-b-5. In some embodiments, D is a structureselected from Table 1, 2 or 3. In some embodiments, D is a structureselected from Table 1. In some embodiments, D is a structure selectedfrom Table 2. In some embodiments, D is a structure selected from Table3.

In some embodiments, the two

units of

are the same. In some embodiments, the two

nits of

are different. In some embodiments, the two

units of

are the same. In some embodiments, the two

units of

are different.

Also falling within the scope of this invention are the in vivometabolic products of compound conjugates described herein, e.g.,compounds of formula II. Such products may result for example from theoxidation, reduction, hydrolysis, amidation, esterification, enzymaticcleavage, and the like, of the administered compound. Accordingly, theinvention includes compounds produced by a process comprising contactinga provided compound, e.g., a compound of formulae I-a, I-b, II, or III,with a subject for a period of time sufficient to yield a metabolicproduct thereof.

In some embodiments, metabolite products are identified by preparing aradiolabelled (e.g. ¹⁴C or ³H) compound, administering it parenterallyin a detectable dose (e.g. greater than about 0.5 mg/kg) to a subjectsuch as a rat, mouse, guinea pig, monkey, or a human being, allowingsufficient time for metabolism to occur (typically about 30 seconds to30 hours), and isolating its conversion products from the urine, bloodor other biological samples. The metabolite structures are determined inconventional fashion, e.g. by MS, LC/MS or NMR analysis. In general,analysis of metabolites is done in the same way as conventional drugmetabolism studies well-known to those skilled in the art. In someembodiments, conversion products, for example, those not otherwise foundin vivo, are useful in diagnostic assays for therapeutic dosing of aprovided compound such as an ADC having the structure of formula II.

In some embodiments, a provided compound generates reactive oxygenspecies (ROS). Exemplary ROS includes superoxide radical anion, hydroxylradical and hydrogen peroxide. In some embodiments, the presentinvention provides a method for generating reactive oxygen species in asubject, comprising providing a compound of formula I-a, I-b, I-c, I-d,II, or III. In some embodiments, a provided compound conjugates withand/or inhibits cellular proteins by forming mixed disulfides betweencysteine residues. In some embodiments, a provided compound conjugateswith and/or inhibits cellular proteins by catalytic formation ofintramolecular disulfide bonds between cysteine residues. In someembodiments, the present invention provides a method for conjugatingwith and/or inhibiting cellular proteins, comprising providing acompound of formula I-a, I-b, I-c, I-d, II, or III. In some embodiments,a provided compound disrupts tertiary structure of proteins containing aZn²⁺-binding cysteine-histidine rich protein domain. In someembodiments, the present invention provides a method for disruptingtertiary structures of proteins containing a Zn²⁺-bindingcysteine-histidine rich protein domain, comprising providing a compoundof formula I-a, I-b, I-c, I-d, II, or III. In some embodiments, aprovided compound ejects Zn²⁺ ions from a protein. In some embodiments,the present invention provides a method for ejecting a Zn²⁺ ion from aprotein, comprising providing a compound of formula I-a, I-b, I-c, I-d,II, or III. In some embodiments, a provided compound inducescaspase-dependent apoptosis. In some embodiments, the present inventionprovides a method for inducing apoptosis, comprising providing acompound of formula I-a, I-b, I-c, I-d, II, or III. In some embodiments,the present invention provides a method for inducing caspase-dependentapoptosis, comprising providing a compound of formula I-a, I-b, I-c,I-d, II, or III.

In some embodiments, the present invention provides compositions ofprovided compounds or pharmaceutically acceptable salts thereof. In someembodiments, a provided composition comprises an effective amount of aprovided compound, or pharmaceutically acceptable salt thereof, fortreatment of a disease, for example, cancer. In some embodiments, acomposition is a pharmaceutical composition. In some embodiments, aprovided composition is suitable for veterinary or human administration.

A provided composition can be in any form that allows for thecomposition to be administered to a subject. For example, a compositioncan be in the form of a solid, liquid or gas (aerosol). Typical routesof administration include, without limitation, oral, topical,parenteral, sublingual, rectal, vaginal, ocular, intra-tumor, andintranasal. Parenteral administration includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. In some embodiments, a provided compound is administeredparenterally. In some embodiments, a provided compound is administeredparenterally. In some embodiments, a provided composition isadministered intravenously. In some embodiments, a provided compositionis administered intravenously.

Pharmaceutical compositions can be formulated so as to allow a providedcompound to be bioavailable upon administration of the composition to apatient. Compositions can take the form of one or more dosage units,where for example, a tablet can be a single dosage unit, a vial maycontain a single dose for intravenous administration, and a container ofa provided compound in aerosol form can hold a plurality of dosageunits.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of a provided compound or composition, themanner of administration, and the composition employed.

A pharmaceutically acceptable carrier or vehicle can be particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous or particulate, so as to provide an aerosol composition usefulin, e.g., inhalatory administration. When intended for oraladministration, a composition is preferably in solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, a composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When a composition is in the form of a capsule, e.g., a gelatin capsule,it can contain, in addition to materials of the above type, a liquidcarrier such as polyethylene glycol, cyclodextrin or a fatty oil.

A composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

Liquid compositions, whether they are solutions, suspensions or otherlike form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or diglycerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of a provided compound that is effective in the treatment ofa particular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the compositions will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

Provided compositions comprise an effective amount of a providedcompound such that a suitable dosage will be obtained. In someembodiments, this amount is at least about 0.01% of a provided compoundby weight of the composition. When intended for oral administration,this amount can be varied to range from about 0.1% to about 80% byweight of the composition. In one aspect, oral compositions can comprisefrom about 4% to about 50% of a provided compound by weight of thecomposition. In yet another aspect, a provided composition is preparedso that a parenteral dosage unit contains from about 0.01% to about 2%by weight of a provided compound or composition.

For intravenous administration, a provided composition can comprise fromabout 0.01 to about 100 mg of a provided compound per kg of a subject'sbody weight. In one aspect, the composition can include from about 1 toabout 100 mg of a provided compound per kg of a subject's body weight.In another aspect, the amount administered will be in the range fromabout 0.1 to about 25 mg/kg of body weight of a provided compound.

Generally, dosage of a provided compound administered to a patient istypically about 0.001 mg/kg to about 2000 mg/kg of a subject bodyweight. In one aspect, a dosage administered to a patient is betweenabout 0.01 mg/kg to about 10 mg/kg of a subject's body weight, inanother aspect, a dosage administered to a subject is between about 0.1mg/kg and about 250 mg/kg of a subject's body weight, in yet anotheraspect, a dosage administered to a patient is between about 0.1 mg/kgand about 20 mg/kg of a subject's body weight, in yet another aspect adosage administered is between about 0.1 mg/kg to about 10 mg/kg of asubject's body weight, and in yet another aspect, a dosage administeredis between about 1 mg/kg to about 10 mg/kg of a subject's body weight.In some embodiments, a daily dosage might range from about 1 μg/kg to100 mg/kg or more, depending on the factors mentioned above. Anexemplary dosage of ADC to be administered to a patient is in the rangeof about 0.1 to about 10 mg/kg of patient weight.

A provided compound can be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.). Administration can be systemic or local. Various deliverysystems are known, e.g., encapsulation in liposomes, microparticles,microcapsules, capsules, etc., and can be used to administer a providedcompound or composition. In certain embodiments, more than one providedcompound or composition is administered to a patient.

In some embodiments, it is desirable to administer one or more providedcompounds or compositions locally to the area in need of treatment. Thiscan be achieved, for example, and not by way of limitation, by localinfusion during surgery; topical application, e.g., in conjunction witha wound dressing after surgery; by injection; by means of a catheter; bymeans of a suppository; or by means of an implant, the implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas silastic membranes, or fibers. In one embodiment, administration canbe by direct injection at the site (or former site) of a cancer, tumoror neoplastic or pre-neoplastic tissue. In another embodiment,administration can be by direct injection at the site (or former site)of a manifestation of an autoimmune disease.

In certain embodiments, it can be desirable to introduce one or moreprovided compounds or compositions into the central nervous system byany suitable route, including intraventricular and intrathecalinjection. Intraventricular injection can be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

In some embodiments, a provided compound or compositions can bedelivered in a controlled release system, such as but not limited to, apump or various polymeric materials can be used. In yet anotherembodiment, a controlled-release system can be placed in proximity of atarget of a provided compound or compositions, e.g., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)). Other controlled-release systems discussed in the review byLanger (Science 249:1527-1533 (1990)) can be used.

In some embodiments, a carrier is a diluent, adjuvant or excipient, withwhich a provided compound is administered. Such pharmaceutical carrierscan be liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents can be used. In one embodiment, whenadministered to a patient, provided compounds or compositions andpharmaceutically acceptable carriers are sterile. Water is an exemplarycarrier when a provided compound is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents.

Provided compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

In some embodiments, a provided compound or composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to a subject particularly a humanbeing. In some embodiments, carriers or vehicles for intravenousadministration are sterile isotonic aqueous buffer solutions. Wherenecessary, a provided composition can also include a solubilizing agent.Compositions for intravenous administration can optionally comprise alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherea provided compound is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where a provided compound isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.In some embodiments, where in tablet or pill form, a providedcomposition can be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compounds. For example, in these later platforms, fluidfrom the environment surrounding a capsule is imbibed by a drivingcompound, which swells to displace an agent or agent composition throughan aperture. In some embodiments, a delivery platform can provide anessentially zero order delivery profile as opposed to the spikedprofiles of immediate release formulations. A time-delay material suchas glycerol monostearate or glycerol stearate can also be used.

A provided composition can be intended for topical administration, inwhich case the carrier may be in the form of a solution, emulsion,ointment or gel base. If intended for transdermal administration, thecomposition can be in the form of a transdermal patch or aniontophoresis device. Topical formulations can comprise a concentrationof a provided compound of from about 0.05% to about 50% w/v (weight perunit volume of composition), in another aspect, from 0.1% to 10% w/v.

A provided composition can be intended for rectal administration, in theform, e.g., of a suppository which will melt in the rectum and release aprovided compound.

A provided composition can include various materials that modify thephysical form of a solid or liquid dosage unit. For example, a providedcomposition can include materials that form a coating shell around theactive ingredients. The materials that form the coating shell aretypically inert, and can be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientscan be encased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. In some embodiments, an aerosol is used todenote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery can be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients.

Whether in solid, liquid or gaseous form, a provided composition caninclude a pharmacological agent used in the treatment of cancer, anautoimmune disease or an infectious disease.

Provided compounds and compositions are useful for treating variousdiseases, for example, cancer, an autoimmune disease or an infectiousdisease in a patient.

In some embodiments, a provided compound is an antibody-drug conjugate(ADC). Antibody-drug conjugates, e.g., a compound of formula II whereinM is an antibody or fragment thereof, may be administered by any routeappropriate to the condition to be treated. In some embodiments, an ADCis administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural.

In some embodiments, provided pharmaceutical formulations of providedantibody-drug conjugates are typically prepared for parenteraladministration, i.e. bolus, intravenous, intratumor injection with apharmaceutically acceptable parenteral vehicle and in a unit dosageinjectable form. In some embodiments, an antibody-drug conjugate havingthe desired degree of purity is optionally mixed with pharmaceuticallyacceptable diluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation or an aqueous solution.

A provided pharmaceutical composition of ADC may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations may be packaged in unit-dose or multi-dose containers, forexample sealed ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water, for injection immediately prior touse. Extemporaneous injection solutions and suspensions are preparedfrom sterile powders, granules and tablets of the kind previouslydescribed. Preferred unit dosage formulations are those containing adaily dose or unit daily sub-dose, as herein above recited, or anappropriate fraction thereof, of the active ingredient.

In some embodiments, the present invention provides veterinarycompositions comprising at least one active ingredient as above definedtogether with a veterinary carrier therefore. Veterinary carriers arematerials useful for the purpose of administering the composition andmay be solid, liquid or gaseous materials which are otherwise inert oracceptable in the veterinary art and are compatible with the activeingredient. These veterinary compositions may be administeredparenterally, orally or by any other desired route.

A provided compound or composition of the present invention may be usedto treat various diseases or disorders, e.g. characterized by theoverexpression of an antigen such as a cancer antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia and lymphoid malignancies. Others include neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologic,including autoimmune, disorders.

In some embodiments, the present invention provides a method for killingor inhibiting proliferation of cells comprising treating the cells withan amount of a provided compound, or a pharmaceutically acceptable saltthereof, being effective to kill or inhibit proliferation of the cells.In some embodiments, the cells are tumor cells or cancer cells. In someembodiments, the present invention provides a method of treating adisease, comprising administering to a subject in need an effectiveamount of a provided compound or a pharmaceutically acceptable saltthereof. In some embodiments, the present invention provides a method oftreating a disease, comprising administering to a subject sufferingtherefrom or susceptible thereto an effective amount of a providedcompound or pharmaceutically salt thereof. In some embodiments, adisease is a cancer, autoimmune disease or infectious disease. In someembodiments, a disease is cancer. In some embodiments, a disease is anautoimmune disease. In some embodiments, a disease is an infectiousdisease. In some embodiments, a provided is a compound of formula I-a.In some embodiments, a provided is a compound of formula I-b. In someembodiments, a provided is a compound of formula II.

A provided compound of the invention may be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, witha second compound having therapeutic properties. A second compound ofthe pharmaceutical combination formulation or dosing regimen preferablyhas complementary activities to a provided compound of the combinationsuch that they do not adversely affect each other.

In some embodiments, a second compound is a chemotherapeutic agent,cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal, adrug for an autoimmune disease, a drug for an infectious disease, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

A combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active agents simultaneously exert their biological activities.

Suitable dosages for co-administered agents are those presently used andmay be lowered due to the combined action (synergy) of the newlyidentified agent and other chemotherapeutic agents or treatments.

A provided combination therapy may provide “synergy” and prove“synergistic”, i.e. the effect achieved when the active ingredients usedtogether is greater than the sum of the effects that results from usingthe compounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

In some embodiments, the present invention provides methods of treatingcancer. In some embodiments, the present invention provides a method oftreating cancer in a subject suffering therefrom, comprisingadministering to the subject a therapeutically effective amount of aprovided compound. In some embodiments, a provided compound has thestructure of formula I-a. In some embodiments, a provided compound hasthe structure of formula I-b. In some embodiments, a provided compoundhas the structure of formula II. In some embodiments, a providedcompound has the structure of formula II-a. In some embodiments, aprovided compound has the structure of formula II-b.

Provided compounds and/or compositions are useful for inhibiting themultiplication of a tumor cell or cancer cell, causing apoptosis in atumor or cancer cell, or for treating cancer in a subject. Providedcompounds and compositions can be used in a variety of settings for thetreatment of cancers. A provided conjugate compound, e.g., a compound offormula II, can be used to deliver a drug to a tumor cell or cancercell. Without being bound by theory, in one embodiment, the ligand unit(M) of a provided compound binds to or associates with a cancer-cell ora tumor-cell-associated antigen, and a provided compound can be taken upinside a tumor cell or cancer cell through receptor-mediatedendocytosis. An antigen can be attached to a tumor cell or cancer cellor can be an extracellular matrix protein associated with the tumor cellor cancer cell. In some embodiments, once inside the cell, a conjugatecompound is cleaved, for example, one or more specific peptide sequenceswithin a linker unit (L) are hydrolytically cleaved by one or moretumor-cell or cancer-cell-associated proteases, resulting in release ofa drug comprising part or all of the drug unit and optionally part orall of the linker unit. A released drug is then free to migrate withinthe cell and induce cytotoxic or cytostatic activities. In some otherembodiments, a provided conjugate compound is cleaved outside a tumorcell or cancer cell, and a drug or drug-linker compound subsequentlypenetrates the cell.

In some embodiments, a ligand unit binds to a tumor cell or cancer cell.In some embodiments, a ligand unit binds to a tumor cell or cancer cellantigen which is on the surface of the tumor cell or cancer cell. Insome embodiments, a ligand unit binds to a tumor cell or cancer cellantigen which is an extracellular matrix protein associated with a tumorcell or cancer cell.

In some embodiments, the specificity of a ligand unit for a particulartumor cell or cancer cell can be important for determining those tumorsor cancers that are most effectively treated. For example, a providedconjugate compound having a BR96 Ligand unit can be useful for treatingantigen positive carcinomas including those of the lung, breast, colon,ovaries, and pancreas. In some embodiments, a provided conjugatecompound having an Anti-CD30 or an anti-CD40 Ligand unit can be usefulfor treating hematologic malignancies.

Other particular types of cancers that can be treated with providedcompounds and/or compositions include, but are not limited to, thoselisted below: Solid tumors, including but not limited to: fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular cancer, small cell lung carcinoma, bladder carcinoma,lung cancer, epithelial carcinoma, glioma, glioblastoma ultiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma. Blood-borne cancers,including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma, acute and chronic leukemias,lymphoblastic, myelogenous, lymphocytic and myelocytic leukemias.Lymphomas: Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,Waldenström's macroglobulinemia, Heavy chain disease, and Polycythemiavera.

In some embodiments, a cancer being treated is carcinoma, lymphoma,blastoma, sarcoma, leukemia or lymphoid malignancies. More particularexamples of such cancers include squamous cell cancer (e.g. epithelialsquamous cell cancer), lung cancer including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

In some embodiments, a provided conjugate compound providesconjugation-specific tumor or cancer targeting, thus reducing generaltoxicity of these compounds. In some embodiments, a linker unitstabilizes a provided compound in blood, yet are cleavable bytumor-specific proteases within the cell, liberating a drug unitoptionally comprising part of the linker unit.

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of a provided compound orcomposition. In some embodiments, a provided compound or composition isadministered with another cancer treatment.

In some embodiments, methods for treating or preventing cancer areprovided, comprising administering to a subject in need thereof aneffective amount of a provided compound or composition. In someembodiments, a provided compound is administered prior to, concurrentlywith, or subsequent to, a chemotherapeutic agent. In some embodiments, achemotherapeutic agent is that with which treatment of the cancer hasnot been found to be refractory. In some embodiments, a chemotherapeuticagent is that with which the treatment of cancer has been found to berefractory. In some embodiments, a provided compound is administered toa patient that has also undergone surgery as treatment for the cancer.

In some embodiments, an additional method of treatment is radiationtherapy. In some embodiments, a provided compound or composition isadministered prior to, concurrently with or subsequent to radiation.

In some embodiments, a provided compound or composition is administeredconcurrently with a chemotherapeutic agent or with radiation therapy. Insome embodiments, a chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of a providedcompound or composition. In some embodiments, a chemotherapeutic agentor radiation therapy is administered concurrently with administration ofa provided compound or composition. In some embodiments, a providedcompound or composition is administered at least one hour, five hours,12 hours, a day, a week, a month, or several months (e.g., up to threemonths), prior or subsequent to administration of a provided compound orcomposition.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents can beadministered. Exemplary chemotherapy drugs are widely known in the art,including but not limited to tubulin-binding drugs, kinase inhibitors,alkylating agents, DNA topoisomerase inhibitors, anti-folates,pyrimidine analogs, purine analogs, DNA antimetabolites, hormonaltherapies, retinoids/deltoids, photodynamic therapies, cytokines,angiogenesis inhibitors, histone modifying enzyme inhibitors, andantimitotic agents. Examples are extensively described in the art,including but not limited to those in PCT Application Publication No.WO2010/025272. In some embodiments, a “tubulin-binding drug” refers to aligand of tubulin or to a compound capable of binding α or β-tubulinmonomers or oligomers thereof, αβ-tubulin heterodimers or oligomersthereof, or polymerized microtubules. Exemplary tubulin-binding drugsinclude, but are not limited to:

a) Combretastatins or other stilbene analogs (e.g., described in Pettitet al, Can. J. Chern., 1982; Pettit et al, J. Org. Chern., 1985; Pettitet al, J. Nat. Prod., 1987; Lin et al, Biochemistry, 1989; Singh et al,J. Org. Chem., 1989; Cushman et al, J. Med. Chem., 1991; Getahun et al,J. Med. Chem., 1992; Andres et al, Bioorg. Med. Chem. Lett., 1993;Mannila, Liebigs. Ann. Chern., 1993; Shirai et al, Bioorg. Med. Chern.Lett., 1994; Medarde et al., Bioorg. Med. Chem. Lett., 1995; Pettit etal, J. Med. Chern., 1995; Wood et al, Br. J. Cancer., 1995; Bedford etal, Bioorg. Med. Chern. Lett., 1996, Dorr et al, Invest. New Drugs,1996; Jonnalagadda et al., Bioorg. Med. Chem. Lett., 1996; Shirai et al,Heterocycles, 1997; Aleksandrzak K, Anticancer Drugs, 1998; Chen et al,Biochem. Pharmacal., 1998; Ducki et al, Bioorg. Med. Chem. Lett., 1998;Hatanaka et al, Bioorg. Med. Chern. Lett., 1998; Medarde, Eur. J. Med.Chem., 1998; Medina et al, Bioorg. Med. Chern. Lett., 1998; Ohsumi etal, Bioorg. Med. Chem. Lett., 1998; Ohsumi et al., J. Med. Chern., 1998;Pettit G R et al., J. Med. Chern., 1998; Shirai et al, Bioorg. Med.Chern. Lett., 1998; Banwell et al, Aust. J. Chern., 1999; Medarde et al,Bioorg. Med. Chern. Lett., 1999; Shan et al, PNAS, 1999; Combeau et al,Mol. Pharmacal, 2000; Pettit et al, J. Med Chem, 2000; Pettit et al,Anticancer Drug Design, 2000; Pinney et al, Bioorg. Med. Chem. Lett.,2000; Flynn et al., Bioorg. Med. Chern. Lett., 2001; Gwaltney et al,Bioorg. Med. Chem. Lett., 2001; Lawrence et al, 2001; Nguyen-Hai et al,Bioorg. Med. Chern. Lett., 2001; Xia et al, J. Med. Chern., 2001; Tahiret al., Cancer Res., 2001; Wu-Wong et al., Cancer Res., 2001; Janik etal, Biooorg. Med. Chem. Lett., 2002; Kim et al., Bioorg Med Chern Lett.,2002; Li et al, Biooorg. Med. Chern. Lett., 2002; Nam et al, Bioorg.Med. Chem. Lett., 2002; Wang et al, J. Med. Chem. 2002; Hsieh et al,Biooorg. Med. Chem. Lett., 2003; Hadimani et al., Bioorg. Med. Chern.Lett., 2003; Mu et al, J. Med. Chern, 2003; Nam, Curr. Med. Chem., 2003;Pettit et al, J. Med. Chern., 2003; WO 02/50007, WO 02/22626, WO02/14329, WO 01/81355, WO 01/12579, WO 01/09103, WO 01/81288, WO01/84929, WO 00/48591, WO 00/48590, WO 00/73264, WO 00/06556, WO00/35865, WO 00/48590, WO 99/51246, WO 99/34788, WO 99/35150, WO99/48495, WO 92/16486, U.S. Pat. Nos. 6,433,012, 6,201,001, 6,150,407,6,169,104, 5,731,353, 5,674,906, 5,569,786, 5,561,122, 5,430,062,5,409,953, 5,525,632, 4,996,237 and 4,940,726 and U.S. patentapplication Ser. No. 10/281,528);b) 2,3-substituted Benzo[b]thiophenes (e.g., described in Pinney et al,Bioorg. Med. Chem. Lett., 1999; Chen et al, J. Org. Chem., 2000; U.S.Pat. Nos. 5,886,025; 6,162,930, and 6,350,777; WO 98/39323);c) 2,3-disubstituted Benzo[b]furans (e.g., described in WO 98/39323, WO02/060872);d) Disubstituted Indoles (e.g., described in Gastpar R, J. Med. Chem.,1998; Bacher et al, Cancer Res., 2001; Flynn et al, Bioorg. Med. Chern.Lett, 2001; WO 99/51224, WO 01/19794, WO 01/92224, WO 01/22954; WO02/060872, WO 02/12228, WO 02/22576, and U.S. Pat. No. 6,232,327);e) 2-Aroylindoles (e.g., described in Mahboobi et al, J. Med. Chem.,2001; Gastpar et al., J. Med. Chern., 1998; WO 01/82909);f) 2,3-disubstituted Dihydronaphthalenes (e.g., described in WO01/68654, WO 02/060872);g) Benzamidazoles (e.g., described in WO 00/41669);h) Chalcones (e.g., described in Lawrence et at, Anti-Cancer Drug Des,2000; WO 02/47604);i) Colchicine, Allocolchicine, Thiocolcichine, Halichondrin B, andColchicine derivatives (e.g., described in WO 99/02166, WO 00/40529, WO02/04434, WO 02/08213, U.S. Pat. Nos. 5,423,753, 6,423,753) inparticular the N-acetyl colchinol prodrug, ZD-6126;j) Curacin A and its derivatives (e.g., described in Gerwick et al, J.Org. Chern., 1994, Blokhin et al, Mol. Phamacol., 1995; Verdier-Pinard,Arch. Biochem. Biophys., 1999; WO 02/06267);k) Dolastatins such as Dolastatin-10, Dolastatin-15, and their analogs(e.g., described in Pettit et al, J. Am. Chern. Soc., 1987; Bai et al,Mol. Pharmacal, 1995; Pettit et al, Anti-Cancer Drug Des., 1998; Poncet,Curr. Pharm. Design, 1999; WO 99/35164; WO 01/40268; U.S. Pat. No.5,985,837);l) Epothilones such as Epothilones A, B, C, D, and Desoxyepothilones Aand B, Fludelone (e.g., described in Chou et al. Cancer Res.65:9445-9454, 2005, the entirety of which is hereby incorporated byreference), 9,10-dehydro-desoxyepothilone B (dehydelone),iso-oxazole-dehydelone (17-isooxazole-dehydelone), fludelone,iso-oxazolefludelone (17-isooxazole-fludelone), (Danishefsky, et al.,PNAS, v. 105, 35:13157-62, 2008; WO 99/02514, U.S. Pat. No. 6,262,094,Nicolau et al., Nature, 1997, Pub. No. US2005/0 143429);m) Inadones (e.g., described in Leoni et al., J. Natl. Cancer Inst.,2000; U.S. Pat. No. 6,162,810);n) Lavendustin A and its derivatives (Mu F et al, J. Med. Chern., 2003,the entirety of which is hereby incorporated by reference);o) 2-Methoxyestradiol and its derivatives (e.g., described in Fotsis etal, Nature, 1994; Schumacher et al, Clin. Cancer Res., 1999; Cushman etal, J. Med. Chem., 1997; Verdier-Pinard et al, Mol. Pharmacal, 2000;Wang et al, J. Med. Chern., 2000; WO 95/04535, WO 01/30803, WO 00/26229,WO 02/42319 and U.S. Pat. Nos. 6,528,676, 6,271,220, 5,892,069,5,661,143, and 5,504,074);p) Monotetrahydrofurans (e.g., “COBRAs”; Uckun, Bioorg. Med. Chern.Lett., 2000; U.S. Pat. No. 6,329,420);q) Phenylhistin and its derivatives (e.g., described in Kanoh et al, J.Antibiot., 1999; Kano et al, Bioorg. Med. Chem., 1999 and U.S. Pat. No.6,358,957);r) Podophyllotoxins such as Epidophyllotoxin (e.g., described inHammonds et al, J. Med. Microbial, 1996; Coretese et al, J. Biol. Chem.,1977);s) Rhizoxins (e.g., described in Nakada et al, Tetrahedron Lett., 1993;Boger et al, J. Org. Chern., 1992; Rao, et al, Tetrahedron Lett., 1992;Kobayashi et al, Pure Appl. Chern., 1992; Kobayashi et al, Indian J.Chern., 1993; Rao et al, Tetrahedron Lett., 1993);t) 2-strylquinazolin-4(3H)-ones (e.g., “SQOs”, Jiang et al, J. Med.Chern., 1990, the entirety of which is hereby incorporated byreference);u) Spongistatin and Synthetic spiroketal pyrans (e.g., “SPIKETs”; Pettitet al, J. Org. Chern., 1993; Uckun et al, Bioorgn. Med. Chern. Lett.,2000; U.S. Pat. No. 6,335,364, WO00/00514);v) Taxanes such as Paclitaxel (TAXOL®), Docetaxel (TAXOTERE®), andPaclitaxel derivatives (e.g., described in U.S. Pat. No. 5,646,176, WIPOPublication No. WO 94/14787, Kingston, J. Nat. Prod., 1990; Schiff etal, Nature, 1979; Swindell et al, J. Cell Biol., 1981);x) Vinca Alkaloids such as Vinblastine, Vincristine, Vindesine,Vinflunine, Vinorelbine (NAVELBINE®) (e.g., described in Owellen et al,Cancer Res., 1976; Lavielle et al, J. Med. Chern., 1991; Holwell et al,Br. J. Cancer., 2001); andy) Welwistatin (e.g., described in Zhang et al, Molecular Pharmacology,1996, the entirety of which is hereby incorporated by reference).

Exemplary specific examples of tubulin-binding drugs include, but arenot limited to, allocolchicine, amphethinile, chelidonine, colchicide,colchicine, combrestatin AI, combretastin A4, combretastain A4phosphate, combrestatin 3, combrestatin 4, cryptophycin, curacin A,deo-dolastatin 10, desoxyepothilone A, desoxyepothilone B,dihydroxypentamethoxyflananone, docetaxel, dolastatin 10, dolastatin 15,epidophyllotoxin, epothilone A, epothilone B, epothilone C, epothiloneD, etoposide, 9,10-dehydro-desoxyepothilone B (dehydelone),iso-oxazole-dehydelone (17-isooxazole-dehydelone), fludelone,iso-oxazolefludelone (17-isooxazole-fludelone), griseofulvin,halichondrin B, isocolchicine, lavendustin A,methyl-3,5-diiodo-4-(4′-methoxyphenoxy)benzoate, N-acetylcolchinol,N-acetylcolchinol-0-phosphate,N-[2-[(4-hydroxyphenyl)amino]-3-pyridyl]-4-methoxybenzenesulfonamide,nocodazole, paclitaxel, phenstatin, phenylhistin, piceid,podophyllotoxin, resveratrol, rhizoxin, sanguinarine, spongistatin 1,steganacin, TAXOL, teniposide, thiocolchicine, vincristine, vinblastine,welwistatin, (Z)-2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenylamine, (Z)-3,5,4′-trimethoxystilbene (R3),2-aryl-1,8-naphthyridin-4(1H)-one, 2-(41-methoxyphenyl)-3-(3 1,4 1,51-trimethoxybenzoyl)-6-methoxybenzo[b]thiophene, 2-methoxy estradiol,2-strylquinazolin-4(3H)-one, 5,6-dihydroindolo(2, 1-a)isoquinoline, and1 0-deacetylbaccatin III.

In some other embodiments, exemplary chemotherapy drugs include but arenot limited to nitrogen mustards, nitrosoureas, alkylsulphonates,triazenes, platinum complexes, epipodophyllins, mitomycins, DHFRinhibitors, IMP dehydrogenase inhibitors, ribonucleotide reductaseinhibitors, uracil analogs, cytosine analogs, purine analogs, receptorantagonists (for example, anti-estrogen, LHRH agonists, anti-androgens),vitamin derivative or analogs, isoprenylation inhibitors, dopaminergicneurotoxins, cell cycle inhibitors, actinomycins, bleomycins,anthracyclines, MDR inhibitors, Ca²⁺ ATPase inhibitors, andanti-metastatis agents. In some embodiments, exemplary specific examplesof tubulin-binding drugs include, but are not limited to,Cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Carmustine,Lomustine, Busulfan, Treosulfan, Dacarbazine, Procarbazine,Temozolomide, Cisplatin, Carboplatin, Aroplatin, Oxaliplatin, Topotecan,Irinotecan, 9-aminocamptothecin, Camptothecin, Crisnatol, Mitomycin C,Methotrexate, Trimetrexate, Mycophenolic acid, Tiazofurin, Ribavirin,5-Ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR),Hydroxyurea, Deferoxamine, 5-Fluorouracil, Fluoxuridine, Doxifluridine,Ralitrexed, Cytarabine, Cytosine arabinoside, Fludarabine, Gemcitabine,Capecitabine, Mercaptopurine, Thioguanine, O-6-benzylguanine, 3-HP,2′-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,ara-C, 5-aza-2′-deoxycytidine, beta-TGDR, cyclocytidine, guanazole,inosine glycodialdehyde, macebecin II, Pyrazoloimidazole, Tamoxifen,Raloxifene, Megestrol, Goserelin, Leuprolide acetate, Flutamide,Bicalutamide, Cis-retinoic acid, All-trans retinoic acid (ATRA-IV), EB1089, CB 1093, KH 1060, Vertoporfin, Phthalocyanine, PhotosensitizerPc4, Demethoxy-hypocrellin A, ABT-627, Bay 12-9566, Benefin, BMS-275291,cartilage-derived inhibitor, CAI, CEP-7055, Col 3, Halofuginone, Heparinhexasaccharide fragment, IM-862, Marimastat, Metalloproteinaseinhibitors, 2-Methoxyestra diol, MMI 270, Neovastat, NM-3, Panzem,PI-88, Placental ribonuclease inhibitor, Plasminogen activatorinhibitor, Prinomastat, Retinoids, Solimastat, Squalamine, SS 3304, SU5416, SU 6668, SU 11248, Tetrahydrocortisol-S, Tetrathiomolybdate,Thalidomide, TNP-470, ZD 6126, ZD 6474, farnesyl transferase inhibitors,Bisphosphonates, trityl cysteine, 1-methyl-4-phenylpyridinium ion,Staurosporine, Actinomycin D, Dactinomycin, Bleomycin A2, Bleomycin B2,Peplomycin, Daunorubicin, Doxorubicin, Idarubicin, Epirubicin,Pirarubicin, Zorubicin, Mitoxantrone, Verapamil, Ardeemin, Ningalin,Thapsigargin, Metastatin, GLiY-SD-ME-1, Sorafenib, Imatinib, Gefinitib,Lapatinib, Dasatinib, Nilotinib, Temsirolimus, Erlotinib, Pomalidomide,Regorafenib, Paclitaxel Protein-Bound Particles For InjectableSuspension, Everolimus, Bosutinib, Cabozantinib, Cabozantinib,Ponatinib, Axitinib, Carfilzomib, Ingenol Mebutate, Regorafenib,Fentanyl, Omacetaxine Mepesuccinate, Cephalotaxine, Pazopanib,Enzalutamide, Fentanyl Citrate, Sunitinib, Vandetanib, Crizotinib,Vemurafenib, Abiraterone Acetate, Eribulin Mesylate, Cabazitaxel,Ondansetron, Pralatrexate, Romidepsin, Plerixafor, Granisetron,Bendamustine Hydrochloride, Raloxifene Hydrochloride, Topotecan,Ixabepilone, Nilotinib, Temsirolimus, Lapatinib, Nelarabine, Sorafenib,Clofarabine, Cinacalcet, Erlotinib, Palonosetron, Tositumomab,Aprepitant, Gefitinib, Abarelix, Conjugated Estrogens, Alfuzosin,Bortezomib, Leucovorin, Fulvestrant, Ibritumomab Tiuxetan, ZoledronicAcid, Triptorelin Pamoate, Arsenic Trioxide, Aromasin, Busulfan,Amifostine, Temozolomide, Odansetron, Dolasetron, Irinotecan,Gemcitabine, Porfimer Sodium, Valrubicin, Capecitabine, Zofran,Bromfenac, Letrozole, Leuprolide, Samarium (¹⁵³sm) Lexidronam,Pamidronate, Anastrozole, Levoleucovorin, Flutamide And Goserelin.

In some embodiments, a provided compound or composition is administeredprior to, concurrently with or subsequent to another polypeptide orprotein. In some embodiments, a polypeptide or protein is a recombinantpolypeptide or protein. Exemplary polypeptides or proteins include butare not limited to cytokines, interferon alfa-2b, interleukin 2,filgrastim, rasburicase, secretin, asparaginase Erwinia chrysanthemi,and ziv-aflibercept. In some embodiments, a polypeptide or proteincomprises an antibody or a fragment of an antibody. In some embodiments,a polypeptide or protein is an antibody or a fragment of an antibody.Examples include but are not limited to rituximab, trastuzumab,tositumomab, alemtuzumab, bevacizumab, cetuximab, panitumumab,ofatumumab, denosumab, ipilimumab, pertuzumab. In some embodiments, apolypeptide or protein is chemically modified. In some embodiments, apolypeptide or protein is conjugated to a drug. In some embodiments, anantibody or an antibody fragment is conjugated to a payload drug,forming an antibody-drug conjugate. In some embodiments, a payload drugis cytotoxic. Exemplary antibody-drug conjugates include but are notlimited to gemtuzumab ozogamicin, brentuximab vedotin, andado-trastuzumab emtansine. In some embodiments, a cancer treatmentcomprises the use of a vaccine. Exemplary vaccines for cancer treatmentare well known in the art, for example but not limited to sipuleucel-T.

A provided compound may be combined with an anti-hormonal compound;e.g., an anti-estrogen compound such as tamoxifen; an anti-progesteronesuch as onapristone (EP 616812); or an anti-androgen such as flutamide,in dosages known for such molecules. Where the cancer to be treated ishormone independent cancer, the patient may previously have beensubjected to anti-hormonal therapy and, after the cancer becomes hormoneindependent, a provided compound (and optionally other agents asdescribed herein) may be administered to the patient. In someembodiments, it may be beneficial to also co-administer acardioprotectant (to prevent or reduce myocardial dysfunction associatedwith the therapy) or one or more cytokines to the patient. In additionto the above therapeutic regimes, the patient may be subjected tosurgical removal of cancer cells and/or radiation therapy.

With respect to radiation, any radiation therapy protocol can be useddepending upon the type of cancer to be treated. For example, but not byway of limitation, X-ray radiation can be administered; in someembodiments, high-energy megavoltage (radiation of greater that 1 MeVenergy) can be used for deep tumors, and electron beam and orthovoltagex-ray radiation can be used for skin cancers. Gamma-ray emittingradioisotopes, such as radioactive isotopes of radium, cobalt and otherelements, can also be administered.

In some embodiments, methods of treatment of cancer with a providedcompound or composition are provided as an alternative to chemotherapyor radiation therapy where the chemotherapy or the radiation therapy hasproven or can prove too toxic, e.g., results in unacceptable orunbearable side effects, for a subject being treated. A subject beingtreated can, optionally, be treated with another cancer treatment suchas surgery, radiation therapy or chemotherapy, depending on whichtreatment is found to be acceptable or bearable.

In some embodiments, a provided compound or composition can be used inan in vitro or ex vivo fashion, such as for the treatment of certaincancers, including, but not limited to leukemias and lymphomas. In someembodiments, such a treatment involves autologous stem cell transplants.In some embodiments, this can involve a multi-step process in which asubject's autologous hematopoietic stem cells are harvested and purgedof all cancer cells, a subject's remaining bone-marrow cell populationis then eradicated via the administration of a high dose of a providedcompound or composition with or without accompanying high dose radiationtherapy, and the stem cell graft is infused back into the animal.Supportive care is then provided while bone marrow function is restoredand a subject recovers.

In some embodiments, the present invention provides methods for treatingan autoimmune disease, comprising administering to a subject sufferingtherefrom or susceptible thereto an effective amount of a providedcompound or a pharmaceutically acceptable salt thereof. In someembodiments, a subject is suffering from an autoimmune disease. In someembodiments, a provided compound is useful for killing or inhibitingreplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. A provided compound or composition canbe used in a variety of settings for the treatment of an autoimmunedisease in a patient. A provided compound can be used to deliver a Drugto a target cell. Without being bound by theory, in some embodiments, aprovided conjugate compound associates with an antigen on the surface ofa target cell, and a provided conjugate compound is then taken up insidea target-cell through receptor-mediated endocytosis. Once inside thecell, a provided conjugate compound can be cleaved. In some embodiments,one or more specific peptide sequences within the linker unit areenzymatically or hydrolytically cleaved, resulting in release of a drugcomprising all or part of the drug unit and optionally part or all ofthe linker unit. A released drug is then free to migrate in the cytosoland induce cytotoxic or cytostatic activities. In an alternativeembodiment, a conjugate compound is cleaved and a drug is releasedoutside the target cell, and the drug subsequently penetrates the cell.

In some embodiments, a ligand unit binds to an autoimmune antigen. Insome embodiments, an antigen is on the surface of a cell involved in anautoimmune condition. In some embodiments, a ligand unit binds to anautoimmune antigen which is on the surface of a cell. In someembodiments, a ligand binds to activated lymphocytes that are associatedwith the autoimmune disease state. In some embodiments, a providedcompound kills or inhibits the multiplication of cells that produce anautoimmune antibody associated with a particular autoimmune disease.

Exemplary types of autoimmune diseases that can be treated with providedcompounds or compositions include, but are not limited to, Th2lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte-relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes); and those listed below:

Active Chronic Hepatitis, Addison's Disease, Allergic Alveolitis,Allergic Reaction, Allergic Rhinitis, Alport's Syndrome, Anaphlaxis,Ankylosing Spondylitis, Anti-phospholipid Syndrome, Arthritis,Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, AtropicRhinitis, Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma,Caplan's Syndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease,Chronic Glomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease,Congenital Rubella Infection, CREST Syndrome, Crohn's Disease,Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis, Discoid Lupus,Dressler's Syndrome, Eaton-Lambert Syndrome, Echovirus Infection,Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr VirusInfection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I). Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia,and Wegener's Granulomatosis.

In some embodiments, an autoimmune disease being treated is selectedfrom rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupusnephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, for example, inflammatory bowel diseases (e.g., ulcerative colitisand Crohn's disease), autoimmune gastritis and pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, and celiac disease), vasculitis (such as, for example,ANCA-associated vasculitis, including Churg-Strauss vasculitis,Wegener's granulomatosis, and polyarteriitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

In some embodiments, the present invention provides methods for treatingan autoimmune disease, comprising administering to a subject sufferingtherefrom an effective amount of a provided compound or composition. Insome embodiments, a provided method comprises administering an effectiveamount of a provided compound or composition and another therapeuticagent known for treatment of an autoimmune disease. Exemplarytherapeutic agents are widely known in the art, including but notlimited to cyclosporine, cyclosporine A, mycophenylate mofetil,sirolimus, tacrolimus, enanercept, prednisone, azathioprine,methotrexate cyclophosphamide, prednisone, aminocaproic acid,chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone,chlorambucil, DHEA, danazol, bromocriptine, meloxicam and infliximab.

In some embodiments, the present invention provides methods for treatingan infectious disease, comprising administering to a subject sufferingtherefrom or susceptible thereto an effective amount of a providedcompound or a pharmaceutically acceptable salt thereof. In someembodiments, a provided compound or composition is useful for killing orinhibiting the multiplication of a cell that produces an infectiousdisease or for treating an infectious disease. A provided compound canbe used in a variety of settings for the treatment of an infectiousdisease in a subject. In some embodiments, a provided conjugate compoundis used to deliver a drug to a target cell. In one embodiment, a ligandunit binds to the infectious disease cell. In one embodiment, a providedcompound kills or inhibits the multiplication of cells that produce aparticular infectious disease.

Exemplary types of infectious diseases that can be treated with aprovided compound include, but are not limited to: Bacterial Diseasessuch as Diphtheria, Pertussis, Occult Bacteremia, Urinary TractInfection, Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis,Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo, Ecthyma,Pneumonia, Endocarditis, Septic Arthritis, Pneumococcal, Peritonitis,Bactermia, Meningitis, Acute Purulent Meningitis, Urethritis,Cervicitis, Proctitis, Pharyngitis, Salpingitis, Epididymitis,Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis, Salmonella,Typhoid Fever, Dysentery, Conjunctivitis, Sinusitis, Brucellosis,Tullaremia, Cholera, Bubonic Plague, Tetanus, Necrotizing Enteritis,Actinomycosis, Mixed Anaerobic Infections, Syphilis, Relapsing Fever,Leptospirosis, Lyme Disease, Rat Bite Fever, Tuberculosis,Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia, Trachoma andInclusion Conjunctivitis: Systemic Fungal Diseases such asHistoplamosis, Coccidiodomycosis, Blastomycosis, Sporotrichosis,Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis,Mycetoma and Chromomycosis; Rickettsial Diseases such as Typhus, RockyMountain Spotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses,Rickettsialpox, Q Fever and Bartonellosis; Parasitic Diseases such asMalaria, Babesiosis, African Sleeping Sickness, Chagas' Disease,Leishmaniasis, Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis,Keratitis, Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis,Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection,Hookworm Infection, Threadworm Infection, Ocular Larva Migrans,Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, RiverBlindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch,Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis,Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Diseaseand Alveolar Hydatid Disease; Viral Diseases such as Measles, Subacutesclerosing panencephalitis, Common Cold, Mumps, Rubella, Roseola, FifthDisease, Chickenpox, Respiratory syncytial virus infection, Croup,Bronchiolitis, Infectious Mononucleosis, Poliomyelitis, Herpangina,Hand-Foot-and-Mouth Disease, Bornholm Disease, Genital Herpes, GenitalWarts, Aseptic Meningitis, Myocarditis, Pericarditis, Gastroenteritis,Acquired Immunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus(HIV), Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral“Walking” Pneumonia, Acute Febrile Respiratory Disease, Acutepharyngoconjunctival fever, Epidemic keratoconjunctivitis, HerpesSimplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles,Cytomegalic Inclusion Disease, Rabies, Progressive MultifocalLeukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-JakobDisease, Gerstmann-Straussler-Scheinker Disease, Tropical SpasticParaparesis, Western Equine Encephalitis, California Encephalitis, St.Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis,Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, MarburgVirus Infections, Ebola Virus Infections and Smallpox.

In some embodiments, the present invention provides methods for treatingan infectious disease, comprising administering to a subject sufferingtherefrom an effective amount of a provided compound or composition. Insome embodiments, a provided method comprises administering an effectiveamount of a provided compound or composition and another therapeuticagent known for treatment of an infectious disease.

In some embodiments, a provided method for treating an infectiousdisease includes administering to a patient in need thereof a providedcompound and another therapeutic agent that is an anti-infectiousdisease agent. Exemplary anti-infectious disease agents are widely knownin the art, including but not limited to β-Lactam Antibiotics such asPenicillin G, Penicillin V, Cloxacilliin, Dicloxacillin, Methicillin,Nafcillin, Oxacillin, Ampicillin, moxicillin, Bacampicillin, Azlocillin,Carbenicillin, Mezlocillin, Piperacillin and Ticarcillin;Aminoglycosides: Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin,Streptomycin and Tobramycin; Macrolides such as Azithromycin,Clarithromycin, Erythromycin, Lincomycin and Clindamycin; Tetracyclinessuch as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline andTetracycline; Quinolones such as Cinoxacin and Nalidixic Acid;Fluoroquinolones such as Ciprofloxacin, Enoxacin, Grepafloxacin,Levofloxacin, Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin andTrovafloxicin; Polypeptides such as Bacitracin, Colistin and PolymyxinB; Sulfonamides such as Sulfisoxazole, Sulfamethoxazole, Sulfadiazine,Sulfamethizole and Sulfacetamide; Miscellaneous Antibacterial Agentssuch as Trimethoprim, Sulfamethazole, Chloramphenicol, Vancomycin,Metronidazole, Quinupristin, Dalfopristin, Rifampin, Spectinomycin,Nitrofurantoin; General Antiviral Agents such as Idoxuradine,Vidarabine, Trifluridine, Acyclovir, Famcicyclovir, Pencicyclovir,Valacyclovir, Gancicyclovir, Foscarnet, Ribavirin, Amantadine,Rimantadine, Cidofovir, Antisense Oligonucleotides, Immunoglobulins andInteferons; Drugs for HIV infection such as Tenofovir, Emtricitabine,Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Nevirapine,Delavirdine, Saquinavir, Ritonavir and Indinavir, Nelfinavir.

Conditions

Suitable conditions for performing provided methods or preparingprovided compounds generally employ one or more solvents. In certainembodiments, one or more organic solvents are used. Examples of suchorganic solvents include, but are not limited to, hydrocarbons such asbenzene, toluene, and pentane, halogenated hydrocarbons such asdichloromethane and chloroform, or polar aprotic solvents, such asethereal solvents including ether, tetrahydrofuran (THF), or dioxanes,or protic solvents, such as alcohols, or mixtures thereof. In someembodiments, a solvent is substituted hydrocarbons. In some embodiments,a solvent is MeNO₂. In some embodiments, a solvent is EtNO₂. In certainembodiments, one or more solvents are deuterated. In some embodiments, asingle solvent is used. In certain embodiments, a solvent is benzene. Incertain embodiments, a solvent is ether. In some embodiments, a solventis a nitrile. In some embodiments, a solvent is acetonitrile.

In some embodiments, mixtures of two or more solvents are used, and insome cases may be preferred to a single solvent. In certain embodiments,the solvent mixture is a mixture of an ethereal solvent and ahydrocarbon. Exemplary such mixtures include, for instance, anether/benzene mixture. Solvent mixtures may be comprised of equalvolumes of each solvent or may contain one solvent in excess of theother solvent or solvents. In certain embodiments wherein a solventmixture is comprised of two solvents, the solvents may be present in aratio of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. Incertain embodiments wherein a solvent mixture comprises an etherealsolvent and a hydrocarbon, the solvents may be present in a ratio ofabout 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1,about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1 etherealsolvent: hydrocarbon. In certain embodiments, the solvent mixturecomprises a mixture of ether and benzene in a ratio of about 5:1. One ofskill in the art would appreciate that other solvent mixtures and/orratios are contemplated herein, that the selection of such other solventmixtures and/or ratios will depend on the solubility of species presentin the reaction (e.g., substrates, additives, etc.), and thatexperimentation required to optimized the solvent mixture and/or ratiowould be routine in the art and not undue.

In some embodiments, a solvent is water. In some embodiments, a solventis water. In some embodiments, a mixture of water with one or more othersolvents is used.

Suitable conditions, in some embodiments, employ ambient temperatures.In some embodiments, a suitable temperature is about 15° C., about 20°C., about 25° C., or about 30° C. In some embodiments, a suitabletemperature is from about 15° C. to about 25° C. In certain embodiments,a suitable temperature is about 20° C., 21° C., 22° C., 23° C., 24° C.,or 25° C.

In certain embodiments, a provided method is performed at elevatedtemperature. In some embodiments, a suitable temperature is from about25° C. to about 110° C. In certain embodiments, a suitable temperatureis from about 40° C. to about 100° C., from about 50° C. to about 100°C., from about 60° C. to about 100° C., from about 70° C. to about 100°C., from about 80° C. to about 100° C., or from about 90° C. to about100° C. In some embodiments, a suitable temperature is about 80° C. Insome embodiments, a suitable temperature is about 30° C. In someembodiments, a suitable temperature is about 40° C. In some embodiments,a suitable temperature is about 50° C. In some embodiments, a suitabletemperature is about 60° C. In some embodiments, a suitable temperatureis about 70° C. In some embodiments, a suitable temperature is about 80°C. In some embodiments, a suitable temperature is about 90° C. In someembodiments, a suitable temperature is about 100° C. In someembodiments, a suitable temperature is about 110° C.

In certain embodiments, a provided method is performed at temperaturelower than ambient temperatures. In some embodiments, a suitabletemperature is from about −100° C. to about 10° C. In certainembodiments, a suitable temperature is from about −80° C. to about 0° C.In certain embodiments, a suitable temperature is from about −70° C. toabout 10° C. In certain embodiments, a suitable temperature is fromabout −60° C. to about 10° C. In certain embodiments, a suitabletemperature is from about −50° C. to about 10° C. In certainembodiments, a suitable temperature is from about −40° C. to about 10°C. In certain embodiments, a suitable temperature is or from about −30°C. to about 10° C. In some embodiments, a suitable temperature is below0° C. In some embodiments, a suitable temperature is about −100° C. Insome embodiments, a suitable temperature is about −90° C. In someembodiments, a suitable temperature is about −80° C. In someembodiments, a suitable temperature is about −70° C. In someembodiments, a suitable temperature is about −60° C. In someembodiments, a suitable temperature is about −50° C. In someembodiments, a suitable temperature is about −40° C. In someembodiments, a suitable temperature is about −30° C. In someembodiments, a suitable temperature is about −20° C. In someembodiments, a suitable temperature is about −10° C. In someembodiments, a suitable temperature is about 0° C. In some embodiments,a suitable temperature is about 10° C.

In some embodiments, a provided method is performed at differenttemperatures. In some embodiments, temperature changes in a providedmethod. In some embodiments, a provided method involves temperatureincrease from a lower suitable temperature to a higher suitabletemperature. In some embodiments, a provided method comprisestemperature increase from about −80° C., about −70° C., about −60° C.,about −50° C., about −40° C., about −30° C., about −20° C., about −10°C., and about 0° C. to about 0° C., about 10° C., about 20° C., ambienttemperature, about 22° C., about 25° C., about 30° C., about 40° C.,about 50° C., about 60° C., about 70° C., about 80° C., about 90° C.,about 100° C., and about 110° C. In some embodiments, a provided methodcomprises temperature increase from about −30° C. to 22° C. In someembodiments, a provided method comprises temperature decrease from ahigher suitable temperature to a lower suitable temperature. In someembodiments, a provided method comprises temperature increase from about110° C., about 100° C., about 90° C., about 80° C., about 70° C., about60° C., about 50° C., about 40° C., about 30° C., about 25° C., about22° C., ambient temperature, about 20° C., about 10° C., and about 0° C.to about 0° C., about −10° C., about −20° C., about −30° C., about −40°C., about −50° C., about −60° C., about −70° C., about −80° C., about−90° C., and about −100° C.

Suitable conditions typically involve reaction times of about 1 minuteto about one or more days. In some embodiments, the reaction time rangesfrom about 0.5 hour to about 20 hours. In some embodiments, the reactiontime ranges from about 0.5 hour to about 15 hours. In some embodiments,the reaction time ranges from about 1.0 hour to about 12 hours. In someembodiments, the reaction time ranges from about 1 hour to about 10hours. In some embodiments, the reaction time ranges from about 1 hourto about 8 hours. In some embodiments, the reaction time ranges fromabout 1 hour to about 6 hours. In some embodiments, the reaction timeranges from about 1 hour to about 4 hours. In some embodiments, thereaction time ranges from about 1 hour to about 2 hours. In someembodiments, the reaction time ranges from about 2 hours to about 8hours. In some embodiments, the reaction time ranges from about 2 hoursto about 4 hours. In some embodiments, the reaction time ranges fromabout 2 hours to about 3 hours. In certain embodiments, the reactiontime is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, 24, 48, 96 or120 hours. In certain embodiments, the reaction time is about 1 hour. Incertain embodiments, the reaction time is about 2 hours. In certainembodiments, the reaction time is about 3 hours. In certain embodiments,the reaction time is about 4 hours. In certain embodiments, the reactiontime is about 5 hours. In certain embodiments, the reaction time isabout 6 hours. In some embodiments, the reaction time is about 12 hours.In some embodiments, the reaction time is about 24 hours. In someembodiments, the reaction time is about 48 hours. In some embodiments,the reaction time is about 96 hours. In some embodiments, the reactiontime is about 120 hours. In certain embodiments, the reaction time isless than about 1 hour. In certain embodiments, the reaction time isabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes. In someembodiments, the reaction time is about 5 minutes. In some embodiments,the reaction time is about 10 minutes. In some embodiments, the reactiontime is about 15 minutes. In some embodiments, the reaction time isabout 20 minutes. In some embodiments, the reaction time is about 25minutes. In some embodiments, the reaction time is about 30 minutes. Insome embodiments, the reaction time is about 35 minutes. In someembodiments, the reaction time is about 40 minutes. In some embodiments,the reaction time is about 100 minutes. In some embodiments, thereaction time is about 110 minutes. In some embodiments, the reactiontime is about 200 minutes. In some embodiments, the reaction time isabout 300 minutes. In some embodiments, the reaction time is about 400minutes.

Some embodiments provide the ability to selectively synthesize productshaving a Z or E configuration about a double bond. In some embodiments,a method of the present invention provides the ability to synthesizecompounds comprising a Z-olefin. In some embodiments, such methods areuseful when applied to a wide range of olefin substrates, includingthose having sterically small or large groups adjacent the olefin. Insome embodiments, the substrate olefins are terminal olefins. In someembodiments, a provided method produces a double bond in a Z:E ratiogreater than about 1:1, greater than about 2:1, greater than about 3:1,greater than about 4:1, greater than about 5:1, greater than about 6:1,greater than about 7:1, greater than about 8:1, greater than about 9:1,greater than about 95:5, greater than about 96:4, greater than about97:3, greater than about 98:2, or, in some cases, greater than about99:1, as determined using methods described herein (e.g., HPLC or NMR).In some cases, about 100% of the double bond produced has a Zconfiguration. The Z or cis selectivity may also be expressed as apercentage of product formed. In some cases, the product may be greaterthan about 50% Z, greater than about 60% Z, greater than about 70% Z,greater than about 80% Z, greater than about 90%0 Z, greater than about95% Z, greater than about 96% Z, greater than about 97% Z, greater thanabout 98% Z, greater than about 99% Z, or, in some cases, greater thanabout 99.5% Z.

Some embodiments provide the ability to selectively synthesize productshaving a Z or E configuration about a double bond. In some embodiments,a method of the present invention provides the ability to synthesizecompounds comprising an E-olefin. In some embodiments, such methods areuseful when applied to a wide range of olefin substrates, includingthose having sterically small or large groups adjacent the olefin. Insome embodiments, the substrate olefins are terminal olefins. In someembodiments, a provided method produces a double bond in a E:Z ratiogreater than about 1:1, greater than about 2:1, greater than about 3:1,greater than about 4:1, greater than about 5:1, greater than about 6:1,greater than about 7:1, greater than about 8:1, greater than about 9:1,greater than about 95:5, greater than about 96:4, greater than about97:3, greater than about 98:2, or, in some cases, greater than about99:1, as determined using methods described herein (e.g., HPLC or NMR).In some cases, about 100% of the double bond produced has an Econfiguration. The E or trans selectivity may also be expressed as apercentage of product formed. In some cases, the product may be greaterthan about 50% E, greater than about 60% E, greater than about 70% E,greater than about 80% E, greater than about 90% E, greater than about95% E, greater than about 96% E, greater than about 97% E, greater thanabout 98% E, greater than about 99% E, or, in some cases, greater thanabout 99.5% E.

In some embodiments, a provided method requires an amount of a compoundwhich promotes a reaction, such that the loading is from about 0.001 mol% to about 20 mol % of the compound relative to substrate. In certainembodiments, the compound is used in an amount of between about 0.001mol % to about 10 mol %. In certain embodiments, the compound is used inan amount of between about 0.001 mol % to about 6 mol %. In certainembodiments, the compound is used in an amount of between about 0.001mol % to about 5 mol %. In certain embodiments, the compound is used inan amount of between about 0.001 mol % to about 4 mol %. In certainembodiments, the compound is used in an amount of between about 0.001mol % to about 3 mol %. In certain embodiments, the compound is used inan amount of between about 0.001 mol % to about 1 mol %. In certainembodiments, the compound is used in an amount of between about 0.001mol % to about 0.5 mol %. In certain embodiments, the compound is usedin an amount of between about 0.001 mol % to about 0.2 mol %. In certainembodiments, the compound is used in an amount of about 0.001 mol %,0.002 mol %, 0.005 mol %, 0.01 mol %, 0.02 mol %, 0.03 mol %, 0.04 mol%, 0.05 mol %, 0.1 mol %, 0.2 mol %, 0.5 mol %, 1 mol %, 2 mol %, 3 mol%, 4 mol %, 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol %. Insome embodiments, the compound is used in an amount of about 0.0002%mol. In some embodiments, the compound is used in an amount of about0.01% mol. In some embodiments, the compound is used in an amount ofabout 3% mol.

In some embodiments, a method of the present invention requires anamount of solvent such that the concentration of the reaction is betweenabout 0.01 M and about 1 M. In some embodiments, the concentration ofthe reaction is between about 0.01 M and about 0.5 M. In someembodiments, the concentration of the reaction is between about 0.01 Mand about 0.1 M. In some embodiments, the concentration of the reactionis between about 0.01 M and about 0.05 M. In some embodiments, theconcentration of the reaction is about 0.01 M. In some embodiments, theconcentration of the reaction is about 0.02 M. In some embodiments, theconcentration of the reaction is about 0.03 M. In some embodiments, theconcentration of the reaction is about 0.04 M. In some embodiments, theconcentration of the reaction is about 0.05 M. In some embodiments, theconcentration of the reaction is about 0.1 M. In some embodiments, theconcentration of the reaction is about 0.3 M.

In some embodiments, a method of the present invention is performed atambient pressure. In some embodiments, a method of the present inventionis performed at reduced pressure. In some embodiments, a method of thepresent invention is performed at a pressure of less than about 20 torr.In some embodiments, a method of the present invention is performed at apressure of less than about 15 torr. In some embodiments, a method ofthe present invention is performed at a pressure of less than about 10torr. In some embodiments, a method of the present invention isperformed at a pressure of about 9, 8, 7, 6, 5, 4, 3, 2, or 1 torr. Incertain embodiments, a method of the present invention is performed at apressure of about 7 torr. In certain embodiments, a method of thepresent invention is performed at a pressure of about 1 torr.

In some embodiments, a method of the present invention is performed atincreased pressure. In some embodiments, a method of the presentinvention is performed at greater than about 1 atm. In some embodiments,a method of the present invention is performed at greater than about 2atm. In some embodiments, a method of the present invention is performedat greater than about 3 atm. In some embodiments, a method of thepresent invention is performed at greater than about 5 atm. In someembodiments, a method of the present invention is performed at greaterthan about 10 atm. In some embodiments, a method of the presentinvention is performed at about 2 atm. In some embodiments, a method ofthe present invention is performed at about 3 atm. In some embodiments,a method of the present invention is performed at about 5 atm. In someembodiments, a method of the present invention is performed at about 10atm.

In some embodiments, a provided method provides chemoselectivity. Insome embodiments, a desired product is produced in greater than about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97% 98%, 99% or 99.5% selectivity.

In some embodiments, a provided method provides stereoselectivity. Insome embodiments, a desired stereoisomer is produced in greater thanabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, 99% or 99.5% selectivity. In some embodiments, aprovided method provides diastereoselectivity. In some embodiments, aprovided method provides diastereoselectivity. In some embodiments, adesired diastereomer is produced in greater than about 50%, 55%, 60%,65%, 70%, 75%. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%or 99.5% selectivity. In some embodiments, a provided method providesenantioselectivity. In some embodiments, a desired enantiomer isproduced in greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 99.5% selectivity.

It will be appreciated that, in certain embodiments, each variablerecited is as defined above and described in embodiments, herein, bothsingly and in combination.

EXEMPLIFICATION

The present invention recognizes, among other things, that there is acontinuing demand for compounds, compositions and methods for treatingvarious diseases, for example, cancer. In some embodiments, the presentinvention provides such compounds, compositions and methods. In someembodiments, the present invention provides methods and uses for suchcompounds and compositions. Exemplary but non-limiting examples aredescribed herein.

The foregoing has been a description of certain non-limiting embodimentsof the invention. Accordingly, it is to be understood that theembodiments of the invention herein described are merely illustrative ofthe application of the principles of the invention. Reference herein todetails of the illustrated embodiments is not intended to limit thescope of the claims.

The epipolythiodiketopiperazine (ETP) alkaloids are a highly complexclass of compounds. In some embodiments, the present invention providesmethods for flexible and scalable synthesis of ETP alkaloids orthiodiketopiperazines, or derivatives and analogs thereof, for example,a provided compound of formula I-a, I-b, I-c or I-d.

Epipolythiodiketopiperazine (ETP; for reviews onepipolythiodiketopiperazines, see: (a) T. W. Jordan and S. J. Cordiner,Trends Pharmacol. Sci., 1987, 8, 144; (b) P. Waring, R. D. Eichner andA. Müllbacher, Med. Res. Rev., 1988, 8, 499; (c) D. M. Gardiner, P.Waring and B. J. Howlett, Microbiology, 2005, 151, 1021; (d) N. J.Patron, R. F. Waller, A. J. Cozijnsen, D. C. Straney, D. M. Gardiner, W.C. Nierman and B. J. Howlett, BMC Evol. Biol., 2007, 7, 174; (e) R.Huang, X. Zhou, T. Xu, X. Yang and Y. Liu, Chem. Biodiv., 2010, 7, 2809;(f) E. Iwasa, Y. Hamashima and M. Sodeoka, Isr. J. Chem., 2011, 51, 420)alkaloids constitute a large (ca. 120 members) and diverse family ofbiologically active secondary metabolites produced by a number offilamentous fungi including those from the Chaetomium, Leptosphaeria,Aspergillus, Verticillium, Penicillium, and Pithomyces genera. Thesecompounds are characterized by the incorporation of an intramolecularpolysulfide bridge at the α,α′-positions of a cyclo-dipeptide (ordiketopiperazine—DKP). Although mono-, di-, tri-, and tetrasulfidemembers are naturally occurring, the disulfides are the most prevalent(For reviews about pharmacologically active sulfur-containing compounds,see: (a) T. Řezanka, M. Sobotka, J. Spižek and K. Sigler, Anti-Infect.Agents Med. Chem., 2006, 5, 187; (b) C.-S. Jiang, W. E. G. Müller, H. C.Schröder and Y.-W. Guo, Chem. Rev., 2012, 112, 2179). In someembodiments, ETP alkaloids containing one or two ETP rings, orderivatives or analogs thereof, possesses a wide spectrum of biologicalactivities (T. W. Jordan and S. J. Cordiner, Trends Pharmacol. Sci.,1987, 8, 144; C.-J. Zheng, C.-J. Kim, K. S. Bae, Y.-H. Kim and W.-G.Kim, J. Nat. Prod., 2006, 69, 1816), including antibacterial ((a) P.Waring and J. Beaver, Gen. Pharmac., 1996, 27, 1311; (b) A. L. Kung, S.D. Zabludoff, D. S. France, S. J. Freedman, E. A. Tanner, A. Vieira, S.Cornell-Kennon, J. Lee, B. Wang, J. Wang, K. Memmert, H.-U. Naegeli, F.Petersen, M. J. Eck, K. W. Bair, A. W. Wood and D. M. Livingston, CancerCell, 2004, 6, 33; (c) D. M. Vigushin, N. Mirsaidi, G. Brooke, C. Sun,P. Pace, L. Inman, C. J. Moody and R. C. Coombes, Med. Oncol., 2004, 21,21; (d) D. Greiner, T. Bonaldi, R. Eskeland, E. Roemer and A. Imhof,Nat. Chem. Biol., 2005, 1, 143; (e) M. Yanagihara, N. Sasaki-Takahashi,T. Sugahara, S. Yamamoto, M. Shinomi, I. Yamashita, M. Hayashida, B.Yamanoha, A. Numata, T. Yamori and T. Andoh, Cancer Sci., 2005, 96, 816;(f) C. R. Isham, J. D. Tibodeau, W. Jin, R. Xu, M. M. Timm and K. C.Bible, Blood, 2007, 109, 2579; (g) Y. Chen, H. Guo, Z. Du, X.-Z. Liu, Y.Che and X. Ye, Cell Prolif, 2009, 42, 838; (h) Y.-M. Lee, J.-H. Lim, H.Yoon, Y.-S. Chun and J.-W. Park, Hepatology, 2011, 53, 171; (i) F. Liu,Q. Liu, D. Yang, W. B. Bollag, K. Robertson, P. Wu and K. Liu, CancerRes., 2011, 71, 6807; (j) K. Yano, M. Horinaka, T. Yoshida, T. Yasuda,H. Taniguchi, A. E. Goda, M. Wakada, S. Yoshikawa, T. Nakamura, A.Kawauchi, T. Miki and T. Sakai, Int. J. Oncol., 2011, 38, 365; (k) N.Zhang, Y. Chen, R. Jiang, E. Li, X. Chen, Z. Xi, Y. Guo, X. Liu, Y.Zhou, Y. Che and X. Jiang, Autophagy, 2011, 7, 598; (l) H. Chaib, A.Nebbioso, T. Prebet, R. Castellano, S. Garbit, A. Restouin, N. Vey, L.Altucci and Y. Collette, Leukemia, 2012, 26, 662; (m) C. R. Isham, J. D.Tibodeau, A. R. Bossou, J. R. Merchan and K. C. Bible, Br. J. Cancer,2012, 106, 314; (n) M. Takahashi, Y. Takemoto, T. Shimazu, H. Kawasaki,M. Tachibana, Y. Shinkai, M. Takagi, K. Shin-ya, Y. Igarashi, A. Ito andM. Yoshida, J. Antiobiot., 2012, 65, 263), anticancer ((a) P. Waring andJ. Beaver, Gen. Pharmac., 1996, 27, 1311; (b) A. L. Kung, S. D.Zabludoff, D. S. France, S. J. Freedman, E. A. Tanner, A. Vieira, S.Cornell-Kennon, J. Lee, B. Wang, J. Wang, K. Memmert, H.-U. Naegeli, F.Petersen, M. J. Eck, K. W. Bair, A. W. Wood and D. M. Livingston, CancerCell, 2004, 6, 33; (c) D. M. Vigushin, N. Mirsaidi, G. Brooke, C. Sun,P. Pace, L. Inman, C. J. Moody and R. C. Coombes, Med. Oncol., 2004, 21,21; (d) D. Greiner, T. Bonaldi, R. Eskeland, E. Roemer and A. Imhof,Nat. Chem. Biol., 2005, 1, 143; (e) M. Yanagihara, N. Sasaki-Takahashi,T. Sugahara, S. Yamamoto, M. Shinomi, I. Yamashita, M. Hayashida, B.Yamanoha, A. Numata, T. Yamori and T. Andoh, Cancer Sci., 2005, 96, 816;(f) C. R. Isham, J. D. Tibodeau, W. Jin, R. Xu, M. M. Timm and K. C.Bible, Blood, 2007, 109, 2579; (g) Y. Chen, H. Guo, Z. Du, X.-Z. Liu, Y.Che and X. Ye, Cell Prolif., 2009, 42, 838; (h) Y.-M. Lee, J.-H. Lim, H.Yoon, Y.-S. Chun and J.-W. Park, Hepatology, 2011, 53, 171; (i) F. Liu,Q. Liu, D. Yang, W. B. Bollag, K. Robertson, P. Wu and K. Liu, CancerRes., 2011, 71, 6807; (j) K. Yano, M. Horinaka, T. Yoshida, T. Yasuda,H. Taniguchi, A. E. Goda, M. Wakada, S. Yoshikawa, T. Nakamura, A.Kawauchi, T. Miki and T. Sakai, Int. J. Oncol., 2011, 38, 365; (k) N.Zhang, Y. Chen, R. Jiang, E. Li, X. Chen, Z. Xi, Y. Guo, X. Liu, Y.Zhou, Y. Che and X. Jiang, Autophagy, 2011, 7, 598; (l) H. Chaib, A.Nebbioso, T. Prebet, R. Castellano, S. Garbit, A. Restouin, N. Vey, L.Altucci and Y. Collette, Leukemia, 2012, 26, 662; (m) C. R. Isham, J. D.Tibodeau, A. R. Bossou, J. R. Merchan and K. C. Bible, Br. J. Cancer,2012, 106, 314; (n) M. Takahashi, Y. Takemoto, T. Shimazu, H. Kawasaki,M. Tachibana, Y. Shinkai, M. Takagi, K. Shin-ya, Y. Igarashi, A. Ito andM. Yoshida, J. Antiobiot., 2012, 65, 263; (o) C.-S. Jiang and Y.-W. Guo,Mini Rev. Med. Chem., 2011, 11, 728), antiviral (W. A. Rightsel, H. G.Schneider, B. J. Sloan, P. R. Graf, F. A. Miller, Q. R. Bartz, J.Ehrlich and G. J. Dixon, Nature, 1964, 204, 1333; P. A. Miller, K. P.Milstrey and P. W. Trown, Science, 1968, 159, 431), antiparasitic,antifungal ((a) J. J. Coleman, S. Ghosh, 1. Okoli and E. Mylonakis, PLoSONE, 2011, 6, e25321; (b) C. Speth, C. Kupfahl, K. Pfaller, M.Hagleitner, M. Deutinger, R. Würzner, I. Mohsenipour, C. Lass-Flörl andG. Rambach, Mol. Immunol., 2011, 48, 2122), antimalarial,immunosuppressive, immunomodulatory ((a) A. Müllbacher, P. Waring, U.Tiwari-Palni and R. D. Eichner, Molec. Immunol., 1986, 23, 231 (b) H. L.Pahl, B. Krauss, K. Schulze-Osthoff, T. Decker, E. B.-M. Traenckner, M.Vogt, C. Myers, T. Parks, P. Waring, A. Mühlbacher, A. P. Czernilofskyand P. A. Baeuerle, J. Exp. Med., 1996, 183, 1829; (c) S. Nishida, L. S.Yoshida, T. Shimoyama, H. Nunoi, T. Kobayashi and S. Tsunawaki, Infect.Immun., 2005, 73, 235; (d) P. Waring, R. D. Eichner and A. Müllbacher,Med. Res. Rev., 1988, 8, 499; (e) P. Waring and J. Beaver, Gen.Pharmac., 1996, 27, 1311), phytotoxic (M. Soledade, C. Pedras, G.Séguin-Swartz and S. R. Abrams, Phytochem., 1990, 29, 777), nematicidal(J.-Y. Dong, H.-P. He, Y.-M. Shen and K.-Q. Zhang, J. Nat. Prod., 2005,68, 1510), antiplatelet (A. Bertling, S. Niemann, A. Uekötter, W.Fegeler, C. Lass-Flörl, C. von Eiff and B. E. Kehrel, Thromb. Haemost.,2010, 104, 270), and anti-inflammatory effects (E. Iwasa, Y. Hamashimaand M. Sodeoka, Isr. J. Chem., 2011, 51, 420). In some embodiments, aprovided compound is antibacterial. In some embodiments, a providedcompound is anticancer. In some embodiments, a provided compound isantiviral. In some embodiments, a provided compound is antiparasitic. Insome embodiments, a provided compound is antifungal. In someembodiments, a provided compound is antimalarial. In some embodiments, aprovided compound is immunosuppressive. In some embodiments, a providedcompound is immunomodulatory. In some embodiments, a provided compoundis phytotoxic. In some embodiments, a provided compound is nematicidal.In some embodiments, a provided compound is antiplatelet. In someembodiments, a provided compound is anti-inflammatory.

Representative Thiodiketopiperazines

A considerable number of synthetic efforts have been directed toward thesynthesis of ETP compounds (For approaches toepipolythiodiketopiperazines, see: (a) P. W. Trown, Biochem. Biophys.Res. Commun., 1968, 33, 402; (b) T. Hino and T. Sato, Tetrahedron Lett.,1971, 12, 3127; (c) H. Poisel and U. Schmidt, Chem. Ber., 1971, 104,1714; (d) H. Poisel and U. Schmidt, Chem. Ber., 1972, 105, 625; (e) E.Öhler, F. Tataruch and U. Schmidt, Chem. Ber., 1973, 106, 396; (f) H. C.J. Ottenheijm, J. D. M. Herscheid, G. P. C. Kerkhoff and T. F. Spande,J. Org. Chem., 1976, 41, 3433; (g) D. L. Coffen, D. A. Katonak, N. R.Nelson and F. D. Sancilio, J. Org. Chem., 1977, 42, 948; (h) J. D. M.Herscheid, R. J. F. Nivard, M. W. Tijhuis, H. P. H. Scholten and H. C.J. Ottenheijm, J. Org. Chem., 1980, 45, 1885; (i) R. M. Williams, R. W.Armstrong, L. K. Maruyama, J.-S. Dung and O. P. Anderson, J. Am. Chem.Soc., 1985, 107, 3246; (j) C. J. Moody, A. M. Z. Slawin and D. Willows,Org. Biomol. Chem., 2003, 1, 2716; (k) A. E. Aliev, S. T. Hilton, W. B.Motherwell and D. L. Selwood, Tetrahedron Lett., 2006, 47, 2387; (l) L.E. Overman and T. Sato, Org. Lett., 2007, 9, 5267; (m) N. W. Polaske, R.Dubey, G. S. Nichol and B. Olenyuk, Tetrahedron: Asym., 2009, 20, 2742;(n) B. M. Ruff, S. Zhong, M. Nieger and S. Bräse, Org. Biomol. Chem.,2012, 10, 935; (o) K. C. Nicolaou, D. Giguère, S. Totokotsopoulos andY.-P. Sun, Angew. Chem. Int. Ed., 2012, 51, 728; (p) P. Waring, R. D.Eichner and A. Müllbacher, Med. Res. Rev., 1988, 8, 499). However, dueto the synthetic challenges posed by the complex molecular architecture,only very few structures could be made, and even for those that havebeen synthesized, only very limited amounts have been provided.Therefore, although various ETP alkaloids have been assessed in adiverse array of biological tests, the non-uniformity of these studiesprecludes comparative analysis and the inference of meaningfulconclusions. In some embodiments, among other things, the presentinvention recognizes that access to greater quantities of ETP alkaloidsor thiodiketopiperazines and their analogs and derivatives is highlydesired. In some embodiments, the present invention provides methods forsynthesizing ETP alkaloids or thiodiketopiperazines and analogs andderivatives thereof. In some embodiments, the present invention providesmethods for synthesizing ETP alkaloids or thiodiketopiperazines andanalogs and derivatives thereof, wherein the methods produce ETPalkaloids or thiodiketopiperazines or analogs and derivatives thereof inquantities large enough to enable uniform biological studies. In someembodiments, the present invention provides new ETP orthiodiketopiperazine compounds and compositions thereof. In someembodiments, the present invention provides ETP or thiodiketopiperazinecompounds and compositions thereof in quantities enough to enableuniform biological studies. In some embodiments, with the providedcompounds and compositions in large enough quantities, the presentinvention analyzes the structural features of the ETP orthiodiketopiperazine compounds and their analogs and derivatives inrelation to the biological activities. In some embodiments, the presentinvention provides evaluation of one or more of the following structuralfactors in relation to biological activities of ETP orthiodiketopiperazine compounds and derivatives and analogs thereof:polysulfide; the number of sulfur atoms, for example, in thepolysulfide; the stereochemical configurations of the sulfuratedcenters; and dimerization state. In some embodiments, the presentinvention recognizes that investigation of the impact of each of thesestructural features is crucial to elucidating the mode of action ofthese compounds, to designing highly potent structures with suitablephysicochemical and biopharmaceutical properties, and to theirtranslation in vivo in clinical applications (e.g., biological probesand chemotherapeutic agents).

In some embodiments, the present provides a method for optimizing a ETPor thiodiketopiperazine compound or derivative or analog thereof,comprising:

(i) maintaining the polysulfide, or modifying the polysulfide to groupsthat can be converted to polysulfide when administered to a subject;

(ii) maintaining the stereochemistry of the sulfurated centers; and

(iii) optionally introducing an electron-withdrawing group to N1, if N1is present.

In some embodiments, an electron-withdrawing group is R³. In someembodiments, an electron-withdrawing group is —S(O)₂R. In someembodiments, an electron-withdrawing group is —S(O)₂Ph.

Exemplary structurally diverse ETP or thiodiketopiperazine alkaloids(and analogs and derivatives thereof) are depicted below:

Compounds that were differentially substituted at the C3-quaternarystereogenic center were constructed and then elaborated with differenttypes of sulfur motifs. For example, compounds 3-7, 10, and 14-67 wereconcisely and efficiently accessed as described in Schemes E1-1-E1-3 oraccording to experimental procedures previously reported by our group.

endo-Tetracyclic bromide 54, prepared from sarcosine 1-tryptophancyclo-dipeptide (N. Boyer and M. Movassaghi, Chem. Sci., 2012, 3, 1798),was used to access epidithiodiketopiperazines bearing differentC3-substituents (Scheme E1-1). Electrophilic activation using silver(I)tetrafluoroborate in nitroethane and trapping of the transient tertiarybenzylic carbocation with the desired nucleophile (i.e., fluoride,N-TIPS-pyrrole (E. M. Beck, N. P. Grimster, R. Hatley and M. J. Gaunt,J. Am. Chem. Soc., 2006, 128, 2528), anisole, 5-Br—N-TIPS-indole)afforded the C3-substituted endo-tetracycles 59 and 68-70 in high yieldsand excellent levels of regio- and stereoselection (N. Boyer and M.Movassaghi, Chem. Sci., 2012, 3, 1798). Dihydroxylation of 59 and 68-70at the C11-methine and C15-methylene positions was achieved withtetra-n-butylammonium permanganate (n-Bu₄NMnO₄, 4 equiv) indichloromethane to provide the corresponding diols in moderate to goodyields as single diastereomers. The direct double cis-thiolation wasaccomplished in a single step and in good to high yields (47-80%) byexposure of the bis-hemiaminals to trifluoroacetic acid (TFA) inhydrogen sulfide-saturated dichloromethane solution followed by mildaerobic oxidation to access the bridgehead disulfides as β-epimers 26,30-33 and α-epimers 34-35. The relative stereochemistry of the α-epimers26, 30-33 of the epidisulfide bridges has been confirmed by key NOESYcross-peaks on the corresponding bis(thiomethylether). In someembodiments, the diastereoselectivities are consistent with the stericbias imposed by the C3-substituents {β:α ratio=2:1 (C3-F); 4:1(C3-Br); >5:1 (C3-pyrrol-3′-yl); >7:1 (C3-indol-3′-yl); >10:1(C3-p-MeOPh)}.

Reagents and conditions: (a) AgBF₄, DTBMP, EtNO₂, 23° C., 1 h, 90%; (b)N-TIPS-pyrrole, AgBF₄, DTBMP, EtNO₂, 0° C., 1 h, 72%; (c) Anisole,AgBF₄, DTBMP, EtNO₂, 0° C., 1 h, 99%; (d) 5-Br—N-TIPS-indole, AgBF₄,DTBMP, EtNO₂, 0° C., 1 h; 83%; (e) H₂, Pd/C, NEt₃, MeOH-EtOAc (2:3 v/v),23° C., 8 h; Et₃N.3HF, 23° C., 13 h, quant.; (f) n-Bu₄NMnO₄, CH₂Cl₂, 23°C., 2 h, 25-52%; (g) H₂S, TFA-EtNO₂ (2:3 v/v), 0 to 23° C., 4 h; O₂,EtOAc, 23° C., 47-80%; TIPS=triisopropylsilyl;DTBMP=2,6-di-tert-butyl-4-methylpyridine; TFA=trifluoroacetic acid.

As exemplified in Scheme E1-2, a set of compounds with a modified sulfurmotif within the DKP core were prepared. Chemo- and stereoselectivethiolation of diol 56 by treatment with TFA in hydrogensulfide-saturated dichloromethane solution at 0° C. generated thecorresponding thiohemiaminal 48 in 90% yield and in a highlydiastereoselective fashion (>10:1 dr). Masking of both alcohol and thiolgroups as isobutyrates and photoinduced reductive removal of thebenzenesulfonyl group gave 51. The desired degree of sulfidation waseventually accomplished by hydrazinolysis, chemoselectiveS-sulfenylation with chloro(triphenylmethane) sulfane or disulfanefollowed by hafnium triflate-mediated cyclization to afford(+)-12-deoxybionectin A (10) and its epitrithiodiketopiperazine congener29 in 65 and 47% yield (3-steps), respectively. A similar two-stepapproach was employed to access benzenesulfonyl-protected epitri- andepitetrathiodiketopiperazines 27 and 28 in 42% and 44% yield,respectively. Ultimately, reduction of the bridgehead disulfide withNaBH₄ followed by in situ S-methylation afforded (+)-gliocladin B (7)and bis(methylthioether) 39 in high yields.

Reagents and conditions: (a) N₂H₄, THF, 0° C., 1 h; Ph₃CSCl, NEt₃, THF,0° C., 90 min, 81% (2-steps); (b) N₂H₄, THF, 0° C., 1 h; Ph₃CSSCl,Hünig's base, THF, 0° C., 25 min; (c) Hf(OTf)₄, MeCN, 23° C., 15 min,n=0: 80%, n=1: 47% (3-steps); (d) H₂S, TFA-CH₂Cl₂ (1:9 v/v), 0 to 23°C., 2 h, 90%, >10:1 dr; (e) Ph₃CSS_(m)Cl, Hünig's base, THF, 0° C., 25min; Hf(OTf)₄, MeCN, 23° C., 50 min, m=1: 42% (2-steps), m=2: 44%(2-steps); (f) MeI, NaBH₄, Pyr, THF, MeOH, 23° C., 45 min, 80%; (g)BnSH, TFA-EtNO₂ (2:3 v/v), 23° C., 3 h, 80%, 17:3 dr. (h) Boc₂O, DMAP,MeCN, 23° C., 3 h, 69%; (i) H₂S, TFA-EtNO₂ (3:4 v/v), 0 to 23° C., 2 h;O₂, EtOAc, 23° C., 77%, >7:1 dr, (j) Boc₂O, DMAP, CH₂Cl₂, 23° C., 7 h,81%; (k) NaBH₄, THF, MeOH, 23° C., 2 h; MOMCl, NEt₃, 23° C., 5 h, 73%;(l) TFA, CH₂Cl₂, 0 to 23° C., 3 h, 81-91%; (m) hν (350 nm),1,4-dimethoxynaphthalene, ascorbic acid, sodium ascorbate, H₂O-MeCN (1:4v/v), 25° C., 2.5 h, 82%; (n) NaBH₄, THF, MeOH, 23° C., 80 min; MEMCl,NEt₃, 23° C., 12 h, 80% (42) and 19% (47); (o) NaBH₄, THF, MeOH, 23° C.,45 min; (p) TCDI, CH₂Cl₂, 23° C., 22 h, 34% (2-steps); (q) CDI, CH₂Cl₂,23° C., 24 h, 8% (2-steps); (r) CH₂I₂, NaBH₄, Pyr, THF, MeOH, 0 to 23°C., 1 h, 46%; (s) P(OEt)₃, THF, 23° C., 6 h, 63%: (t) AcCl, Pyr, CH₂Cl₂,23° C., 4 h, 63% (2-steps); (u) MeSCl, Pyr, CH₂Cl₂, 0 to 23° C., 2 h,49% (2-steps); (v) (MeS)₂, THF, 23° C., 19 h, 41% (2-steps);TFA=trifluoroacetic acid; Pyr=pyridine; Boc₂O=di-tert-butyl dicarbonate;DMAP=4-(dimethylamino)pyridine; TCDI=1,1′-thiocarbonyldiimidazole;CDI=1,1′-carbonyldiimidazole; MOMCl=chloromethyl methyl ether,MEMCl=2-methoxyethoxymethyl chloride.

(+)-Gliocladin C (52, Y. Usami, J. Yamaguchi and A. Numata,Heterocycles, 2004, 63, 1123) and several C11-hydroxylated (57-58) andC11,C12-dehydrogenated (53) intermediates were prepared following theprocedures previously reported for the synthesis of this atypicalnon-thiolated triketopiperazine (N. Boyer and M. Movassaghi, Chem. Sci.,2012, 3, 1798).

Exposure of hemiaminal 56 to benzyl mercaptan and TFA in nitroethaneresulted in the formation of the corresponding bis(benzylthioether)(C15β:C15α=5.7:1) in 80% yield (single diastereomer, C15β). Furtherderivatization of the indole nitrogen with a t-butoxycarbonyl group gave43 in 69% yield. After masking the indole substituent of the key ETPintermediate 26, the bridgehead disulfide was reduced with NaBH₄ andS-methoxymethylated in a single flask. Subsequent t-butoxycarbonylremoval with TFA in dichloromethane afforded bis(thioether) 40 in 66%yield over two steps. A similar strategy including the photoinducedreductive removal of the N1-benzenesulfonyl group provided 41 in 55%over three steps. Reduction of the sulfur bridge of ETP 24 with NaBH₄ ina mixture of THF and methanol and in situ trapping of the resultingthiolates with 2-methoxyethoxy-methyl chloride (MEMCl) led to thioether47 and bis(thioether) 42 in 19% and 80% yield, respectively.

Further modifications to the sulfur bridge were accomplished bytreatment of the corresponding dithiol (obtained from NaBH₄ reduction ofETP 26) with 1,1′-thiocarbonyldiimidazole (TCDI) or1,1′-carbonyldiimidazole (CDI) to afford di- and trithiocarbonates 36and 37, respectively. Similarly, thioacetal 38 was accessed directly bydouble alkylation using diiodomethane. Desulfurization ofepidithiodiketopiperazine 26 was realized by treatment withtriethylphosphite in THF to give epimonosulfide 25 in 63% yield (F.Cherblanc, Y.-P. Lo, E. De Gussem, L. Alcazar-Fuoli, E. Bignell, Y. He,N. Chapman-Rothe, P. Bultinck, W. A. Herrebout, R. Brown, H. S. Rzepaand M. J. Fuchter, Chem.—Eur. J., 2011, 17, 11868). The sulfur atomswere also capped with the S-acetyl and S-methylsulfane functionalgroupings to afford compounds that are potentially more labile underintracellular conditions. After reduction of the sulfur bridge ofepidisulfide 26, its treatment with an excess of acetyl chloride,methanesulfenyl chloride, or dimethyldisulfide afforded compounds 44, 45and 46, respectively, in good yields.

Compounds with substituent at the C15 position were also prepared, forexample, compounds derived from N-methyl-1-alanine1-tryptophancyclo-dipeptide. As exemplified in Scheme E1-3, synthesis of thesederivatives commenced with endo-tetracyclic bromide 73. Tertiarybenzylic bromide 73 also proved to be an excellent substrate for thedesired regio- and stereoselective Friedel-Crafts-type coupling with5-bromo-1-triisopropylsilylindole (67% over 2 steps) to affordC3-indolyl tetracycle 74. Allylation of the C3-tertiary benzylic halideusing allyltributylstannane under radical conditions (K. M. Depew, S. P.Marsden, D. Zatorska, A. Zatorski, W. G. Bornmann and S. J. Danishefsky,J. Am. Chem. Soc., 1999, 121, 11953) followed by hydrogenation of theterminal olefin afforded C3-n-propyl tetracycle 75. These twoC3-substituted tetracyclic monomers were subsequently subject tohydroxylation conditions using bis(pyridine) silver(I) permanganate(Pyr₂AgMnO₄) in pyridine. Treatment of the resultant diols withpotassium trithiocarbonate and TFA in dichloromethane resulted in rapidformation of the desired monomeric dithiepanethiones 64 and 66 in 63%yield as a 5:1 isomeric mixture, as well as 65 and 67 in 52% and 17%yield, respectively. Exposure of these compounds to ethanolamine inacetone followed by oxidative workup using potassium triiodide yieldedthe corresponding epidithiodiketopiperazine analogs 60-63.

Synthesis of exemplary dimeric DKP and ETP derivatives (Schemes Si andS2) were described below. The diacetate forms of these epidi- andepitrithiodiketopiperazines (15-16) were also synthesized. A variety ofderivatives (14, 18-19, 21-23) possessing the sulfonyl group were alsoprepared.

Certain synthesized compounds were screened for their ability to inducedeath in two human cancer cell lines: U-937 (leukemic monocyte lymphoma)and HeLa (cervical cancer). Compounds that demonstrated anticanceractivity at 1 μM or below were retested in triplicate at a range ofcompound concentrations to generate logistical dose-response curves fromwhich IC₅₀ values were derived. The results are presented in Table E1-1.

TABLE E1-1 Assessment of exemplary ETPs and DKPs for anticancer activityagainst U-937 (hystiocytic lymphoma) and HeLa (cervical carcinoma) humancancer cell lines after a 72-hour exposure.^(a) Cmpd U-937 HeLa Dimerswith epipolysulfide bridges 3 15.5 ± 2.9  7.2 ± 3.0 4 0.81 ± 0.15 6.9 ±2.0 5 0.75 ± 0.13 6.3 ± 0.6 6 1.3 ± 0.5 5.6 ± 1.0 14 0.18 ± 0.06 0.09 ±0.06 15 4.7 ± 1.3 14.1 ± 7.4  16 9.7 ± 2.1 70 ± 20 17 8.9 ± 2.3 28.1 ±2.2  18 2.9 ± 2.1 1.1 ± 0.8 Sulfur-containing dimers 19 >10,000 >10,00020 >10,000 >10,000 Dimers without sulfur 21 >1,000 >10,00022 >1,000 >10,000 23 >1,000 >10,000 Sarcosine-derived monomers withsulfur-containing bridge 10 17.4 ± 1.1  117 ± 26  24 5.9 ± 1.4 75 ± 1025 710 ± 134 1476 ± 182  26 2.8 ± 0.3 41.7 ± 5.7  27 36.6 ± 2.4  27.5 ±9.2  28 8.3 ± 3.3 137 ± 86  29 24.5 ± 7.4  123 ± 29  30 5.0 ± 1.3 44.9 ±1.3  31 20.7 ± 6.4  1530 ± 440  32 14.8 ± 3.1  26.8 ± 4.8  33 5.0 ± 7.122.2 ± 10.9 34 59 ± 23 550 ± 33  35 21.3 ± 8.7  56 ± 21 36 3.8 ± 0.9 33± 13 37 5.5 ± 1.3 37 ± 15 38 82 ± 28 465 ± 37  Sarcosine-derivedmonomers with non-bridging sulfur 7 >10,000 >10,000 39 >10,000 >10,00040 >1,000 >1,000 41 >10,000 >10,000 42 >10,000 >10,00043 >10,000 >10,000 44 4.1 ± 0.5 17.3 ± 4.5  45 14.5 ± 9.1  13.7 ± 2.3 46 20.4 ± 9.0  36.6 ± 3.9  Sarcosine-derived monomers without sulfur47 >10,000 >10,000 48 >10,000 >10,000 49 >10,000 >10,00050 >1,000 >1,000 51 >10,000 >1,000 52 >10,000 >10,000 53 >10,000 >10,00054 >10,000 >10,000 55 >10,000 >10,000 56 >10,000 >10,00057 >10,000 >10,000 58 >10,000 >10,000 59 >10,000 >10,000N-Methylalanine-derived monomers with sulfur-containing bridge 60 56 ±23 67 ± 11 61 >1,000 >1,000 62 >1,000 >1,000 63 >1,000 >1,000 64 24 ± 2 25.3 ± 2.0  65 >1,000 >1,000 66 >1,000 >1,000 67 >1,000 >1,000 ≤1 nM 1 <x ≤ 10 nM 10 < x ≤ 50 nM 50 < x ≤ 150 nM 150 < x ≤ 1,000 nM >1,000 nM^(a)72-hour IC₅₀ values (in nM) as determined by MTS (U-937) and SRB(HeLa). Error is standard deviation of the mean, n ≥ 3.; Cmpd =compound; DKP = diketopiperazine; ETP = epipolythiodiketopiperazine;IC₅₀ = half maximal inhibitory concentration; MTS =3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;SRB = sulforhodamine B.

Among tested compounds, in both U-937 and HeLa cells, the homodimers arethe most potent compounds {IC₅₀ (U-937)≥0.18 nM; IC₅₀ (HeLa)≥0.09 nM},with the N1, N1′-benzenesulfonylated analog (14) of (+)-12,12′-dideoxyverticillin A (3) showing the best activity {IC₅₀ (U-937):0.18 nM; IC₅₀ (HeLa): 0.09 nM}. Monomeric ETP derivatives also show goodactivity in both HeLa (IC₅₀≥5.9 nM) and U-937 (IC₅₀≥2.8 nM) human cancercell lines. Within the N1-benzenesulfonyl monomeric class, variousaromatic substituents (indol-3′-yl 26, N-Boc-indol-3′-yl 24,pyrrol-3′-yl 32, p-MeO-phenyl 33) are well tolerated at the C3-positionand their IC₅₀'s are of the same order of magnitude {IC₅₀ (U-937):2.8-14.8 nM; IC₅₀(HeLa): 22-75 nM}. Halide substitution at C3 (bromide30, fluorides 31 and 35) results in intermediately good activity. Whilenot wishing to be limited by theory, Applicant notes that in someembodiments, the steric environment of the C3 position may be crucialfor biological activity: n-alkyl groups at that position (n-propylanalogs 61 and 65) lead to substantially lower potencies than moresterically hindered (hetero)aryl and halide substituents or the C3′quaternary carbon of a second monomeric subunit, and the dimers in someembodiments have better activity than (hetero)arylated monomers. In someembodiments, dimers containing two sulfur bridge groups are one order ofmagnitude more potent than monomeric C3-(3′-indolyl) analogs and 2 to 3orders of magnitude more potent than heterodimers bearing a singlesulfur bridge. This non-linear increase of biological activity betweenmono- and dimeric ETP compounds was also observed in other families.While not wishing to be limited by theory, Applicant notes that theobservation may suggest a synergistic effect; pharmacokinetic propertiescould also play a role.

Comparing homodimers (+)-12,12′-dideoxyverticillin A (3), (+)-chaetocinA (4), (+)-chaetocin C (5), and (+)-12,12′-dideoxychetracin A (6)head-to-head reveals that, in some embodiments, the chaetocin-type ETPderivatives are more potent than their non-C15-hydroxylated counterparts{IC₅₀ (U-937): 0.75-1.3 nM vs. 15.5 nM; IC₅₀(HeLa): 5.6-6.9 nM vs. 7.2nM}. In some embodiments, acetylation of the 17,17′-hydroxyl groups(15-16) also results in a reduction of potency (5.8- to 12.9-fold forU-937; 2.0- to 11.1-fold for HeLa). Methyl substitution at C15 inmonomeric alkaloids (Trp-Ala cyclo-dipeptides 60 vs. 26 and 64 vs. 36)affects the potency of the compounds moderately in the test. Without theintention to be limited by theory, Applicant notes that the differencein potency between the different types of substituents at C15 may begenerally minimal, although it could be sensitive to variations of thesteric environment. In some embodiments, the present invention providesa method of optimizing an ETP compound or a derivative of an analogthereof, comprising modifying substituents at the C15 position. In someembodiments, a provided method is used to optimize the pharmacokineticparameters during drug development. In some embodiments, a compound offormula I-a, I-b, I-c, or I-d is connected to L through C15 or asubstituent on C15.

In some embodiments, the present invention discovered that substitutionat N1 and N1′ with electron-withdrawing groups, such as benzenesulfonyl(14) or trifluoroacetyl (17) groups, enhanced the anticancer activity ofthe alkaloids. For example, sulfonyl group (14) dramatically increasedthe potency (2 orders of magnitude more potent than the correspondingsecondary aniline (+)-12,12′-dideoxyverticillin A (3)). Thetrifluoroacetamide at N1 and N1′ (17 vs. 16) also enhanced theanticancer activity. For the monomeric ETP-containing analogs, theN1-benzenesulfonyl substitution also amplifies the anticancer effect inU-937 cell line (epidisulfide: 26 vs. 10; epitrisulfide: 27 vs. 29).Without the intention to be limited by theory, Applicant notes that theN1 group could confer significantly higher chemical stability, and mayalso affect the pharmacodynamic properties of these ETP compounds.

In some embodiments, the present invention demonstrated that sulfurationat only the tryptophan C_(α)-position is not sufficient for potentactivity {bis(trisulfanes) (19 and 20), C11-thioesters (50-51),C11-thioether (49), and C11-thiols (47-48)}. In some embodiments, aprovided method for optimizing an ETP compound, or derivatives oranalogs, thereof comprises maintaining or installing sulfurization atboth C_(α)-position of the diketopiperazine ring (e.g., C11 and C15). Insome embodiments, both amino acid C_(α) positions have sulfuration byseparate sulfur atoms.

In some embodiments, sulfur derivatives possessing non-labile alkylgroups {S-methylthioethers (7 and 39), S-(methoxymethyl)thioethers(40-41), S-(2-ethoxyethoxymethyl)thioether (42 and 47),S-benzylthioether (43)} did not display any anticancer activity (IC₅₀>10μM). Thioacetal 38 were active. In some embodiments, the degree ofsulfuration of the polysulfide bridge {dimers: (+)-chaetocin A (4) vs.(+)-chaetocin C (5) vs. (+)-12,12′-dideoxychetracin A (6) or 15 vs. 16;monomers: 10 vs. 29, 26 vs. 27 vs. 28} has no substantial impact on celldeath induction; in some embodiments, the IC₅₀ values are within themargin of error of each other.

In addition to ETP-containing compounds, several monomeric or dimericderivatives provided by the present invention possessing modificationsdirectly on the sulfur bridge are competent anticancer agents. Exemplaryderivative compounds include thioacetate (44), dithiocarbonate (37),trithiocarbonates (18, 36, 64), and alkyl disulfides (45-46). Withoutthe intention to be limited by theory, Applicant notes that certainderivatives could be readily converted to the thiols; for example, themethyl disulfides would readily be converted to the thiols throughreduction or nucleophilic displacement. While not wishing to be limitedby theory, Applicant notes that the data could suggest a mode of actionthat involves a common intermediate; in the presence of a reducingcytoplasmic environment combined with the presence of enzymes—ydrolases,carboxylesterases, and lipases (W. Kroutil, A. A. Stämpfli, R. Dahinden,M. Jörg, U. Müller and J. P. Pachlatko, Tetrahedron, 2002, 58,2589)—methyl disulfides, thioacetates, and thiocarbonates could play therole of prodrugs ((a) C. A. Fink, J. E. Carlson, P. A. McTaggart, Y.Qiao, R. Webb, R. Chatelain, A. Y. Jeng and A. J. Trapani, J. Med.Chem., 1996, 39, 3158; (b) B. Testa and J. M. Mayer, Hydrolysis in Drugand Prodrug Metabolism; Wiley-VCH: Weinheim, 2003; (c) J. Rautio, H.Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Järvinen and J.Savolainen, Nature Rev. Drug Disc., 2008, 7, 255). Without the intentionto be limited by theory, Applicant notes that these compounds may beconverted to their corresponding epidisulfide pharmacophores, whichcould be potentially actively concentrated within the cell via aglutathione-dependent uptake mechanism ((a) P. H. Bernardo, N. Brasch,C. C. L. Chai and P. Waring, J. Biol. Chem., 2003, 278, 46549; and (b)C. S. Sevier and C. A. Kaiser, Nature Rev. Mol. Cell Biol., 2002, 3,836). In some embodiments, compounds with the sulfur bridge on theα-face of the DKP (34, 62-63, 66-67) are inactive.

Certain provided compounds were tested in culture against a panel ofthree supplementary human cancer cell lines representing threeadditional tumor histologies (H460, lung carcinoma; 786-O, renalcarcinoma; MCF-7, breast carcinoma). As shown in Table E1-2, the ETPsretain their potency across the cell lines. In some embodiments, aprovided compound is active in all cell lines. In some embodiments, aprovide compound can be used to selectively target certain cells and/orcancers. For example, compound 14 retains high levels of activity in allof the cell lines, while compound 17 is more active toward some of thecell lines. Without the intention to be limited, Applicant notes thatcertain cell lines could be more sensitive to provided compounds; forexample, U-937 and HeLa are slightly more sensitive to the ETPs than theother three cell lines.

TABLE E1-2 Assessment of exemplary compounds for cytotoxicity in fivehuman cell lines {U-937 (hystiocytic lymphoma), HeLa (cervicalcarcinoma), H460 (lung carcinoma), 786-O (renal carcinoma), and MCF-7(breast carcinoma)} after a 72-hour exposure.^(a) Compound U-937 HeLaH460 786-O MCF-7 Dimers with epipolysulfide bridges 3 15.5 ± 2.9  7.2 ±3.0 42 ± 15 33.5 ± 12.6 28.4 ± 4.5  4 0.81 ± 0.15 6.9 ± 2.0 53 ± 23 26.5± 7.7  50 ± 16 5 0.75 ± 0.13 6.3 ± 0.6 26.0 ± 9.3  14.6 ± 0.8 27 ± 11 61.3 ± 0.5 5.6 ± 1.0 39.3 ± 41   16.0 ± 2.4  22.5 ± 6.0  14 0.18 ± 0.060.09 ± 0.06 1.53 ± 0.85 1.55 ± 0.77 1.65 ± 0.51 15 4.7 ± 1.3 14.1 ± 7.4 140 ± 82  95 ± 23 77 ± 36 16 9.7 ± 2.1 70 ± 20 187 ± 79  204 ± 40  263 ±98  17 8.9 ± 2.3 28.1 ± 2.2  179 ± 88  189 ± 26  >333 18 2.9 ± 2.1 1.1 ±0.8 18.5 ± 2.7  14.9 ± 4.4  10.7 ± 8.1  Sarcosine-derived monomers withsulfur-containing bridge 10 17.4 ± 1.1  117 ± 26  215 ± 100 181 ± 26 156 ± 18  26 2.8 ± 0.3 41.7 ± 5.7  66 ± 19 62.7 ± 7.6  67 ± 19 27 36.6 ±2.4  27.5 ± 9.2  146.4 ± 6.9  145 ± 10  151 ± 31  28 8.3 ± 3.3 137 ± 86 277 ± 43  344 ± 20  309 ± 31  29 24.5 ± 7.4  123 ± 29  348 ± 110 168 ±34  217 ± 35  30 5.0 ± 1.3 44.9 ± 1.3  277 ± 44  162 ± 35  152 ± 42  3120.7 ± 6.4  1530 ± 440  >1000 368 ± 78  218 ± 97  32 14.8 ± 3.1  26.8 ±4.8  31.8 ± 2.5  66 ± 23 61 ± 11 33 5.0 ± 2.1 22.2 ± 10.9 46.7 ± 5.3  83± 18 63 ± 16 35 21.3 ± 8.7  56 ± 21 123 ± 50  64 ± 20 93 ± 34 36 3.8 ±0.9 33 ± 13 190 ± 61  133 ± 10  163 ± 7  37 5.5 ± 1.3 37 ± 15 119 ± 25 139 ± 3  145 ± 8  Sarcosine-derived monomers with non-bridging sulfur 444.1 ± 0.5 17.3 ± 4.5  149 ± 59  151 ± 7  103 ± 37  45 14.5 ± 9.1  13.7 ±2.3  252 ± 37  376 ± 120 373 ± 75  N-Methylalanine-derived monomers withsulfur-containing bridge 60 56 ± 23 67 ± 11 165 ± 65  907 ± 48  327 ±36  64 24 ± 12 25.3 ± 2.0  222 ± 72  266 ± 85  210 ± 73  ≤1 nM 1 < x ≤10 nM 10 < x ≤ 50 nM 50 < x ≤ 150 nM >150 nM ^(a)72-hour IC₅₀ values (innM) as determined by MTS (U-937) and SRB (HeLa, H460, 786-O, and MCF-7).Error is standard deviation of the mean, n ≥ 3; Cmpd = compound; IC₅₀ =half maximal inhibitory concentration; MTS =3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;SRB = sulforhodamine B.

The polysulfide dimers (+)-12,12′-dideoxyverticillin A (3),(+)-chaetocins A (4) and C (5), (+)-12,12′-dideoxychetracin A (6), 14,and bisdithiepanethione 18 are active across the board. In the case ofthe N1,N1′-benzenesulfonylated analog of (+)-12,12′-dideoxyverticillin A(14), the potency is dramatically increased (2 orders of magnitude morepotent than with the addition of the benzenesulfonyl group.

In some embodiments, the degree of sulfuration has a larger impact insome of the adherent cell lines tested (H460, 786-O, and MCF-7),especially in the case of 26-28. In some embodiments, this set ofcompounds shows a 2-fold decrease in activity with each additionalsulfur atom in the polysulfide bridge. Without the intention to belimited by theory, Applicant notes that lack of substitution at N1 (10and 29, and dimers 4-6) mitigates this effect.

Bisthioacetate 44, trithiocarbonate 36 and dithiocarbonate 37 displaylower activity than the corresponding epidisulfide 26, but are activetoward the cell lines. These compounds show consistently lower IC₅₀values than epidisulfide 10 (up to 6.7-fold more potent). In someembodiments, a provided compound displayed different activity acrosscell lines. C3-pyrrolyl 32 and C3-p-methoxyphenyl 33 epidisulfidesdisplayed a consistent potency (4-fold change in activity, as comparedto 6- to 40-fold changes for other derivatives) in addition to goodrelative potency in H460 cells.

The exemplary compounds were evaluated for their ability to inducehemolysis in human erythrocytes at concentrations well above theiranticancer IC50 values. As shown in FIG. 2 the compounds show nohemolytic activity. The concentrations at which hemolytic activity wasassessed (1 and 10 μM) are, in some cases, over 1000-fold higher thanthe IC50 values for cancer cells.

Compounds 5, 14, 26 and 33 were examined for their ability to inducecaspase-dependent apoptotic pathway on U-937 cells. The induction ofapoptosis was first evaluated by the level of phosphatidylserineexternalization (detected by FITC-conjugated Annexin V (AnnV)) thatoccurs prior to the disruption of cell membrane integrity (detected bypropidium iodide (PI)). The progression of cells through the AnnV+/PI−quadrant (lower right, FIG. 1A) demonstrates the ability of bothmonomeric and dimeric ETP-containing derivatives to induce apoptosis.

Another marker of apoptosis is the cleavage patterns of variousintracellular proteins. Caspase-3, one of the key apoptotic executionercaspases, is activated from its low-activity zymogen (procaspase-3) atan early stage of apoptosis. This activation was visualized by Westernblot (FIG. 1B) by the cleavage of procaspase-3 (35 kDa) to caspase-3 (12and 17 kDa). Caspase-3 in turn cleaves one of its cellular substrates,PARP-1. Treating cells with the four ETP derivatives, followed byWestern blotting for procaspase-3/caspase-3 and PARP-1 revealed that allthese compounds induce cleavage of procaspase-3 and PARP-1. Without theintention to be limited by theory, Applicant notes that data in FIGS. 1Ato 1B indicate that these ETP derivatives may induce caspase-dependentapoptic cell death.

The present invention provides methods and compounds that enablestructural-activity relationship study. Some of the results werepresented below in Scheme E1-4.

Without the intention to be limited by theory, Applicant notes that somehuman cancer cell lines were most responsive to variations infunctionalization at the C3 and C11/C15 centers while displaying a moremodest response to modification at the N1 and C17 positions. Again,without the intention to be limited by theory, Applicant notes thatcompounds may be highly potent if the diketopiperazines were sulfuratedat the C11/C15 sites in a manner consistent with a species capable ofbeing converted to an epidisulfide on the same face of thehexahydropyrrolo[1,2-a]pyrazine-1,4-dione moiety as N1 in the biologicalmilieu. In some embodiments, the anticancer potencies of this collectionof compounds correlate positively with the steric environment at the C3position, rendering the dimeric ETP alkaloids, in certain cases, themost potent with (sub)nanomolar IC₅₀'s against a range of human cancercell lines. Without the intention to be limited by theory, Applicantnotes that the muted sensitivity of these cell lines to variations in N1and C17 substitution make these ideal sites for compound optimization.In some embodiments, despite their attenuated activity, a lowermolecular-weight monomers may prove to have more optimal pharmacokineticproperties and provide further avenues for molecular modification in theoptimization and development of small-molecule drugs.

General Procedures.

All reactions were performed in oven-dried or flame-dried round-bottomflasks. The flasks were fitted with rubber septa, and reactions wereconducted under a positive pressure of argon. Cannulae or gas-tightsyringes with stainless steel needles were used to transfer air- ormoisture-sensitive liquids. Where necessary (so noted), solutions weredeoxygenated by sparging with argon for a minimum of 10 min. Flashcolumn chromatography was performed as described by Still et al. usinggranular silica gel (60-Å pore size, 40-63 μm, 4-6% H₂O content,Zeochem) (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,2923). Analytical thin layer chromatography (TLC) was performed usingglass plates pre-coated with 0.25 mm 230-400 mesh silica gel impregnatedwith a fluorescent indicator (254 nm). TLC plates were visualized byexposure to short wave ultraviolet light (254 nm), reversibly stainedwith iodine (I₂ absorbed on silica) vapor, and irreversibly stained bytreatment with an aqueous solution of ceric ammonium molybdate (CAM)followed by heating (˜1 min) on a hot plate (˜250° C.). Organicsolutions were concentrated at 29-30° C. on rotary evaporators capableof achieving a minimum pressure of ˜2 torr. The benzenesulfonylphotodeprotection was accomplished by irradiation in a Rayonet RMR-200photochemical reactor (Southern New England Ultraviolet Company,Branford, Conn., USA) equipped with 16 lamps (RPR-3500, 24 W,λ_(max)=350 nm, bandwidth˜20 nm).

Materials.

Commercial reagents and solvents were used as received with thefollowing exceptions: dichloromethane, acetonitrile, tetrahydrofuran,methanol, pyridine, toluene, and triethylamine were purchased from J. T.Baker (Cycletainer™) and were purified by the method of Grubbs et al.under positive argon pressure (Pangborn, A. B.; Giardello, M. A.;Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15,1518). Nitromethane and nitroethane (from Sigma-Aldrich) were purifiedby fractional distillation over calcium hydride and were stored overLinde 3 Å molecular sieves in Schlenk flasks sealed with septa andTeflon tape under argon atmosphere (Armarego, W. L. F.; Chai, C. L. L.Purification of Laboratory Chemicals, 5^(th) ed.; Butterworth-Heinemann:London, 2003). Hünig's base and benzene were dried by distillation fromcalcium hydride under an inert argon atmosphere and used directly.1,4-Dimethoxynaphthalene, hafnium (IV) trifluoromethanesulfonatehydrate, and iodomethane were purchased from Alfa Aesar;1-(triisopropylsilyl)-1H-pyrrole was purchased from Combi-Block;triphenylmethanesulfenyl chloride was purchased from TCI America, Inc;2,6-di-tert-butyl-4-methylpyridine (DTBMP) was purchased from OChemIncorporation. All other solvents and chemicals were purchased fromSigma-Aldrich. Silver tetrafluoroborate (≥99.99% trace metals basis) andhydrogen sulfide (≥99.5%) were purchased from Sigma-Aldrich.1,4-Dimethoxynaphthalene was purified by crystallization from absoluteethanol.

Instrumentation.

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded with aBruker AVANCE-600 NMR spectrometer (with a Magnex Scientificsuperconducting actively-shielded magnet) or a Varian inverse probe 500INOVA spectrometer, are reported in parts per million on the δ scale,and are referenced from the residual protium in the NMR solvent (CDCl₃:δ 7.26 (CHCl₃), acetone-d₆: δ 2.05 (acetone-ds), acetonitrile-d₃: δ 2.13(acetonitrile-d₂), DMSO-d₆: δ 2.50 (DMSO-ds), methanol-d₄: δ 3.31(methanol-d₃)) (Fulmer, G. R.; Miller, A. J. M.; Sherden, N. H.;Gottlieb, H. E.; Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg,K. I. Organometallics 2010, 29, 2176). Data are reported as follows:chemical shift [multiplicity (br=broad, s=singlet, d=doublet, t=triplet,q=quadruplet, sp=septet, m=multiplet), coupling constant(s) in Hertz,integration, assignment]. Carbon-13 nuclear magnetic resonance (¹³C NMR)spectra were recorded with a Bruker AVANCE-600 NMR spectrometer (with aMagnex Scientific superconducting actively-shielded magnet), a BrukerAVANCE-400 NMR spectrometer (with a Magnex Scientific superconductingmagnet), or a Varian 500 INOVA spectrometer, are reported in parts permillion on the δ scale, and are referenced from the carbon resonances ofthe solvent (CDCl₃: δ 77.23, acetone-d₆: δ 29.84, acetonitrile-d₃: δ118.26, DMSO-d₆: δ 39.52). Data are reported as follows: chemical shift(multiplicity (given if applicable), coupling constant in Hertz (givenif applicable), assignment). Fluorine-19 nuclear magnetic resonance (¹⁹FNMR) spectra were recorded with a Varian Mercury 300 spectrometer, arereported in parts per million on the δ scale, and are referenced fromthe fluorine resonance of neat trichlorofluoromethane (CFCl₃: δ 0). Dataare reported as follows: chemical shift. Infrared data (IR) wereobtained with a Perkin-Elmer 2000 FTIR and are reported as follows:frequency of absorption (cm⁻¹), intensity of absorption (s=strong,m=medium, w=weak, br=broad). Optical Rotations were recorded on a JascoP-1010 Polarimeter (chloroform, Aldrich, Chromasolv Plus 99.9%; acetone,Aldrich, Chromasolv Plus 99.9%) and specific rotations are reported asfollows: [wavelength of light, temperature (° C.), specific rotation,concentration in grams/100 mL of solution, solvent]. Preparative HPLCwas performed on a Waters system with the 1525 Binary HPLC Pump, 2489UV/Vis Detector, 3100 Mass Detector, System Fluidics Organizer, and 2767Sample Manager components. We are grateful to Dr. Li Li and Deborah Bassfor obtaining the mass spectrometric data at the Department ofChemistry's Instrumentation Facility, Massachusetts Institute ofTechnology. High-resolution mass spectra (HRMS) were recorded on aBruker Daltonics APEXIV 4.7 Tesla FT-ICR-MS using an electrospray (ESI)ionization source.

Positional Numbering System.

At least three numbering systems for dimeric diketopiperazine alkaloidsexist in the literature ((a) Von Hauser, D.; Weber, H. P.; Sigg, H. P.Helv. Chim. Acta 1970, 53, 1061. (b) Barrow, C. J.; Cai, P.; Snyder, J.K.; Sedlock, D. M.; Sun, H. H.; Cooper, R. J. Org. Chem. 1993, 58, 6016.(c) Springer, J. P.; Büchi, G.; Kobbe, B.; Demain, A. L.; Clardy, J.Tetrahedron Lett. 1977, 28, 2403). In assigning the ¹H and ¹³C NMR dataof all intermediates en route to our different naturally occurring ETPsand their synthetic analogs, a uniform numbering scheme was to employed.For ease of direct comparison, particularly between early intermediates,non-thiolated diketopiperazines, and advanced compounds, the numberingsystem used by Barrow for (+)-WIN-64821 (using positional numbers 1-21)is optimal and used throughout this report. In key instances, theproducts are accompanied by the numbering system as shown below.

Exemplary C3-substituted epipolythiodiketopiperazines anddiketopiperazines (Certain experimental procedure and characterizationdata are described in ⁹ Boyer, N.; Movassaghi, M. Chem. Sci. 2012, 3,1798).

General Reagents and Methods for Biological Assays.

For biological assays, propidium iodide and phenazine methosulfate werepurchased from Sigma-Aldrich. The3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumsalt was obtained from Promega. Human erythrocytes were purchased fromBioreclamation and used within three days of receipt. Optical densitieswere recorded on a Spectramax Plus 384 (Molecular Devices, Sunnyvale,Calif.). Flow cytometry was performed on a BD Biociences LSR II (SanJose, Calif.) and the data was analyzed as described using FACSDivasoftware (San Jose, Calif.).

Cell Culture Information.

Cells were grown in media supplemented with fetal bovine serum (FBS) andantibiotics (100 μg/mL penicillin and 100 U/mL streptomycin).Specifically, experiments were performed using the following cell linesand media compositions: U-937, HeLa, H460, and 786-O (RPMI-1640+10%FBS), and MCF7 (EMEM+10% FBS). Cells were incubated at 37° C. in a 5%CO₂, 95% humidity atmosphere.

IC₅₀ Value Determination for Adherent Cells Using Sulforhodamine B(SRB).

Adherent cells (HeLa, H460, 786-O, and MCF7) were added into 96-wellplates (5,000 cells/well for HeLa cell line; 2,000 cells/well for H460,786-O, and MCF7 cell lines) in 100 μL media and were allowed to adherefor 2-3 hours. Compounds were solubilized in DMSO as 100× stocks, addeddirectly to the cells (100 μL final volume), and tested over a range ofconcentrations in triplicate (1% DMSO final) on a half-log scale.Concentrations tested ranged from 1 pM to 10 μM, depending on thepotency of the compound. DMSO and cell-free wells served as the live anddead control, respectively. After 72 hours of continuous exposure, theplates were evaluated using the SRB colorimetric assay as describedpreviously (Vichai, V.; Kirtikara, K. Nature Prot. 2006, 1, 1112).Briefly, media was removed from the plate, and cells were fixed by theaddition of 100 μL cold 10% trichloroacetic acid in water. Afterincubating at 4° C. for an hour, the plates were washed in water andallowed to dry. Sulforhodamine B was added as a 0.057% solution in 1%acetic acid (100 μL), and the plates were incubated at room temperaturefor 30 minutes, washed in 1% acetic acid, and allowed to dry. The dyewas solubilized by adding 10 mM Tris base solution (pH 10.5, 200 μL) andincubating at room temperature for 30 minutes. Plates were read at λ=510nm. IC₅₀ values were determined from three or more independentexperiments using TableCurve (San Jose, Calif.).

IC₅₀ Value Determination for Non-Adherent Cells Using MTS.

In a 96-well plate, compounds were pre-added as DMSO stocks intriplicate to achieve a final concentration of 1%. DMSO and cell-freewells served as the live and dead control, respectively. U-937 (5,000cells/well) cells were distributed in 100 μL media to thecompound-containing plate. After 72 hours, cell viability was assessedby adding 20 μL of a PMS/MTS solution (Cory, A. H.; Owen, T. C.;Barltrop, J. A.; Cory, J. G. Cancer Commun. 1991, 3, 207) to each well,allowing the dye to develop at 37° C. until the live control hadprocessed MTS, and reading the absorbance at λ=490 nm. IC₅₀ values weredetermined from three or more independent experiments using TableCurve(San Jose, Calif.).

Hemolysis Assay Using Human Erythrocytes.

To prepare the erythrocytes, 0.1 mL of human blood was centrifuged(10,000 g, 2 min). The pellet was washed three times with saline (0.9%NaCl) via gentle re-suspension and centrifugation (10,000 g, 2 min).Following the final wash, the erythrocytes were re-suspended in 0.8 mLred blood cell (RBC) buffer (10 mM Na₂HPO₄, 150 mM NaCl, 1 mM MgCl₂, pH7.4).

DMSO stocks of compounds were added to 0.5 mL tubes in singlicate (1 μL,3.3% DMSO final). The stocks were diluted with 19 μL RBC buffer.Positive control tubes contained DMSO in water, and negative controltubes contained DMSO in RBC buffer. A suspension of washed erythrocytes(10 μL) was added to each tube, and samples were incubated at 37° C. for2 hours. Samples were centrifuged (10,000 g, 2 min), and the supernatantwas transferred to a clear, sterile 384-well plate. The absorbance ofthe supernatants was measured at λ=540 nm, and percent hemolysis wascalculated relative to the average absorbance values measured for thecontrols.

Apoptosis in U-937 Cells with Annexin V-FITC and Propidium Iodide(AnnV/PI).

DMSO stocks of compounds were added to a 24-well plate in singlicate(0.2% DMSO final). After compound addition, 0.5 mL of a U-937 cellsuspension (250,000 cells/mL) was added and allowed to incubate for 24hours. Following treatment, the cell suspensions were transferred toflow cytometry tubes and pelleted (500 g, 3 min). The media was removedby aspiration, and cells were re-suspended in 200 μL AnnV binding buffer(10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) with 5 μg/mL PI and1:90 dilution of AnnV. Samples were analyzed using flow cytometry.

Apoptosis in U-937 Cells by Western Blot Analysis.

In a 24-well plate, compounds were added as DMSO stocks (0.2% DMSOfinal) in singlicate. After compound addition, 1.5 mL of a U-937 cellsuspension (250,000 cells/mL) was added and allowed to incubate for 24hours. The cell suspensions were transferred to 1.5 mL tubes andpelleted (600 g, 3 min). The media was removed via aspiration, and thecells were lysed by adding 40 μL of RIPA buffer (50 mM Tris, pH 8.0, 150mM NaCl, 1% TX-100, 0.5% sodium deoxycholate, 0.1% SDS) with 1% ProteaseInhibitor Cocktail Set III. Each sample was then vigorously vortexedtwice for 15 seconds, with a 15-minute incubation on ice following eachagitation. The cellular debris was pelleted (16,100 g, 5 min), and then33 μL of the protein suspension was transferred to fresh 0.5 mL tubes.The protein levels were quantified using a standard BCA (ThermoScientific), after which the samples were diluted with deionized waterto achieve equal protein concentrations for all samples.

Prior to analyzing the samples, 6× Laemmli sample buffer (350 mM Tris,pH 6.8, 12% SDS, 0.012% bromophenol blue, 47% glycerol) with 5%β-mercaptoethanol was added to each sample to achieve a final 1×concentration, after which the samples were incubated at 95° C. for 5minutes to denature the protein samples. 20-30 μg of protein was addedto a 15-well 4-20% Tris-HCl gel and run for 1 hour at 120 V. The gel wasequilibrated PBS (pH 7.4) for 5 minutes, and then transferred to a PVDFmembrane for 2 hours at 45 V.

Generally, blots were probed as follows. The blot was blocked overnightat 4° C. with a blocking agent in 0.05% Tris-Buffered Saline Tween-20(TBST) and then probed for the primary antibody at a 1:1000 dilutionwith a blocking agent in TBST overnight at 4° C. The blot was washedwith TBST, and then probed with a secondary rabbit HRP antibody(1:10,000, Cell Signaling) in TBST for 1 hour at room temperature. Theblot was washed with TBST and PBS, and then visualized with Picoluminescent substrate kit (Thermo Scientific). Caspase 3 and PARP wereblocked in 5% milk, and actin was blocked in 5% BSA.

C3-(5-Bromo-1-TIPS-Indol-3′-yl)-Pyrrolidinoindoline (+)-S12:

A round-bottom flask was charged with endo-tetracyclic bromide (+)-54(5.00 g, 10.5 mmol, 1 equiv), 2,6-di-tert-butyl-4-methylpyridine (DTBMP,2.59 g, 12.6 mmol, 1.20 equiv), and5-bromo-1-triisopropylsilyl-1H-indole (S11, 14.8 g, 42.0 mmol, 4.00equiv. 5-Bromo-1-triisopropylsilyl-1H-indole S11 was prepared inquantitative yield by silylation of commercially available 5-bromoindoleusing triisopropylsilyl chloride and sodium hydride in tetrahydrofuran.For preparation and characterization, see: Brown, D. A.; Mishra, M.;Zhang, S.; Biswas, S.; Parrington, I.; Antonio, T.; Reith, M. E. A.;Dutta, A. K. Bioorg. Med. Chem. 2009, 17, 3923), and the mixture wasdried azeotropically (concentration of a benzene solution, 2×30 mL)under reduced pressure and placed under an argon atmosphere. Anhydrousnitroethane (120 mL) was introduced via syringe, and the mixture wascooled to 0° C. in an ice-water bath. A solution of silver(I)tetrafluoroborate (6.30 g, 32.4 mmol, 3.09 equiv) in anhydrousnitroethane (40 mL) at 0° C. was introduced via cannula to the solutioncontaining the tetracyclic bromide (+)-54 over 20 min. After 5 min, awhite precipitate was observed in the clear yellow reaction solution.The reaction flask was covered in aluminum foil, and the suspension wasmaintained at 0° C. After 1 h, saturated aqueous sodium chloridesolution (25 mL) was introduced, and the resulting biphasic mixture wasvigorously stirred for 30 min at 0° C. The reaction mixture was dilutedwith ethyl acetate (150 mL), was filtered through a Celite pad, and thesolid was washed with ethyl acetate (3×50 mL). The combined filtrateswere washed with 5% aqueous citric acid solution (2×100 mL), water(3×100 mL), and saturated aqueous sodium chloride solution (75 mL). Theorganic layer was dried over anhydrous sodium sulfate, was filtered, andwas concentrated under reduced pressure. The resulting orange residuewas purified by flash column chromatography (eluent: gradient, 2→10%acetone in dichloromethane) to afford the indole adduct (+)-S12 (6.56 g,83.6%) as a white foam. Structural assignments were made with additionalinformation from gCOSY, HSQC, gHMBC, and NOESY data. ¹H NMR (600 MHz,CDCl₃, 20° C.): δ 8.04 (app-d, 0.1=7.4, 2H, SO₂Ph-o-H), 7.77 (d, J=8.3,1H, C₈H), 7.56 (app-t, J=7.5, 1H, SO₂Ph-p-H), 7.42 (app-dd, J=7.8, 8.0,2H, SO₂Ph-m-H), 7.30 (d, J=8.9, 1H, C_(8′)H), 7.29 (app-dt, J=1.1, 7.9,1H, C₇H), 7.15 (app-dd, J=1.8, 8.8, 1H, C_(7′)H), 6.98 (app-t, J=7.5,1H, C₆H), 6.94 (s, 1H, C_(2′)H), 6.84 (d, J=7.4, 1H, C₅H), 6.55 (d,J=1.3, 1H, C_(5′)H), 6.28 (s, 1H, C₂H), 4.47 (dd, J=8.0, 9.5, 1H, C₁₁H),4.07 (d, J=17.8, 1H, C₁₅H_(a)), 3.94 (d, J=17.8, 1H, C₁₅H_(b)), 3.03(dd, J=7.6, 13.8, 1H, C₁₂H_(a)), 3.00 (s, 3H, C₁₇H₃), 2.86 (dd, J=10.0,13.9, 1H, C₁₂H_(b)), 1.59 (app-sp, J=7.5, 3H, C_(10′)H), 1.08 (app-d,J=8.5, 18H, C_(11′)H). ¹³C NMR (100 MHz, CDCl₃, 20° C.): δ 167.7 (C₁₃),166.8 (C₁₆), 141.3 (C_(9′)), 139.7 (C₉), 137.1 (SO₂Ph-ipso-C), 134.2(SO₂Ph-p-C), 134.0 (C₄), 130.9 (C_(2′)), 130.3 (C_(4′)), 129.6 (C₇),129.3 (SO₂Ph-m-C), 127.9 (SO₂Ph-o-C), 125.4 (C_(7′)), 124.6 (C₆), 124.0(C₅), 121.9 (C_(5′)), 116.0 (C_(8′)), 115.7 (C₈), 115.1 (C_(3′)), 113.5(C_(6′)), 82.7 (C₂), 59.5 (C₁₁), 55.4 (C₃), 54.6 (C₁₅), 37.6 (C₁₂), 33.8(C₁₇), 18.2 (C_(11′)), 12.9 (C_(10′)). FTIR (thin film) cm⁻¹: 2949 (m),2869 (m), 1681 (s), 1447 (m), 1396 (m), 1366 (m), 1178 (s), 1092 (w),987 (w), 732 (m), 690 (w). HRMS (ESI) (m/z): calc'd for C₃₇H₄₄BrN₄O₄SSi[M+H]⁺: 747.2030, found: 747.2025. [α]_(D) ²⁴: +93.6 (c=0.26, CHCl₃).TLC (10% acetone in dichloromethane), Rf: 0.67 (UV, CAM).

C3-(Indol-3′-yl)-Pyrrolidinoindoline (+)-59:

A mixture of anhydrous methanol and ethyl acetate (3:2 v/v, 160 mL) wasintroduced into a round-bottom flask charged with the indole adduct(+)-S12 (6.56 g, 8.77 mmol, 1 equiv) and palladium on activated charcoal(10% w/w, 0.50 g, 0.47 mmol, 0.05 equiv). The flask was purged by threecycles of vacuum and dihydrogen and sealed under an atmosphere ofhydrogen gas (15 psi). Triethylamine (1.50 mL, 10.7 mmol, 1.22 equiv)was introduced to the flask via syringe, and the resulting suspensionwas vigorously stirred at 23° C. Upon completion of the reaction (ca 8h) as monitored by TLC, the flask was purged by three cycles of vacuumand argon and sealed under argon atmosphere. Neat triethylaminetrihydrofluoride (3.00 mL, 18.4 mmol, 2.15 equiv, McClinton, M. A.Aldrichimica Acta 1995, 28, 31) was introduced to the flask via syringeand the resulting suspension was stirred at 23° C. After 13 h, thereaction mixture was filtered through a pad of Celite. The solids werewashed with ethyl acetate (3×50 mL). The combined filtrates wereconcentrated under reduced pressure. The resulting pale yellow solid wasdiluted in ethyl acetate (400 mL) and washed sequentially with anaqueous hydrochloric acid solution (1 N, 2×100 mL), water (2×100 mL),and saturated aqueous sodium chloride solution (50 mL). The organiclayer was dried over anhydrous sodium sulfate, was filtered, and wasconcentrated under reduced pressure. The resulting residue was purifiedby flash column chromatography (eluent: 15% acetone in dichloromethane)to afford the indole adduct (+)-59 (4.59 g, 99.9%) as a white solid.Structural assignments were made with additional information from gCOSY,HSQC, gHMBC, and NOESY data.

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.03 (br-s, 1H, N_(1′)H), 7.75 (d,J=8.2, 1H, C₈H), 7.50 (d, J=7.6, 2H, SO₂Ph-o-H), 7.38 (t, J=7.5, 1H,SO₂Ph-p-H), 7.35 (d, J=8.2, 1H, C_(8′)H), 7.30 (app-dt, J=1.1, 7.8, 1H,C₇H), 7.19 (app-t, J=7.6, 1H, C_(7′)H), 7.10 (app-t, J=7.9, 2H,SO₂Ph-m-H), 7.09-7.06 (m, 1H, C₅H), 7.06 (app-t, J=7.4, 1H, C₆H), 6.93(app-t, J=7.4, 1H, C_(6′)H), 6.89 (d, J=7.9, 1H, C₅H), 6.37 (s, 1H,C₂H), 6.16 (d, J=2.3, 1H, C_(2′)H), 4.56 (app-t, J=8.1, 1H, C₁₁H), 4.13(d, J=17.5, 1H, C₁₅H_(a)), 3.85 (d, J=17.5, 1H, C₁₅H_(b)), 3.09 (dd,J=8.9, 14.1, 1H, C₁₂H_(a)), 3.03 (dd, J=7.2, 14.1, 1H, C₁₂H_(b)), 2.90(s, 3H, C₁₇H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 167.5 (C₁₃), 165.9(C₁₆), 139.6 (C₉), 137.6 (SO₂Ph-ipso-C), 137.4 (C_(9′)), 135.9 (C₄),133.1 (SO₂Ph-p-C), 129.3 (C₇), 128.6 (SO₂Ph-m-C), 127.6 (SO₂Ph-o-C),125.2 (C₆), 124.8 (C₅), 124.6 (C_(4′)), 123.6 (C_(2′)), 122.9 (C_(7′)),120.3 (C_(6′)), 119.0 (C_(5′)), 117.1 (C₈), 115.0 (C_(3′)), 112.0(C_(8′)), 83.8 (C₂), 58.8 (C₁₁), 55.4 (C₃), 54.6 (C₁₅), 36.1 (C₁₂), 33.8(C₁₇). FTIR (thin film) cm⁻¹: 3384 (br-m), 3013 (w), 2925 (w), 1681 (s),1457 (m), 1399 (m), 1355 (m), 1169 (m), 1091 (w), 751 (m). HRMS (ESI)(m/z): calc'd for C₂₈H₂₄N₄NaO₄S [M+Na]⁺: 535.1410, found: 535.1413.[α]_(D) ²³: +70.0 (c=0.15, CHCl₃). TLC (25% acetone in dichloromethane),Rf: 0.41 (UV, CAM).

C3-(Indol-3′-yl) Hexacyclic Diol (−)-56:

Freshly prepared tetra-n-butylammonium permanganate (Sala, T.; Sargent,M. V. J. Chem. Soc., Chem. Commun. 1978, 253. Tetra-n-butylammoniumpermanganate was prepared according to a literature procedure (Karaman,H.; Barton, R. J.; Robertson, B. E.; Lee, D. G. J. Org. Chem. 1984, 49,4509) and dried under reduced pressure at room temperature. (a) Gardner,K. A.; Mayer, J. M. Science 1995, 269, 1849. (b) Strassner, T.; Houk, K.N. J. Am. Chem. Soc. 2000, 122, 7821. (c) Shi, S.; Wang, Y.; Xu, A.;Wang, H.; Zhu, D.; Roy, S. B.; Jackson, T. A.; Busch, D. H.; Yin, G.Angew. Chem. Int. Ed. 2011, 50, 7321) (767 mg, 2.12 mmol, 3.79 equiv)was added as a solid to a solution of the indole adduct (+)-59 (287 mg,0.56 mmol, 1 equiv) in dichloromethane (20 mL) at 23° C. After 30 min,the dark purple solution was diluted with saturated aqueous sodiumsulfite solution (20 mL) and then with ethyl acetate (160 mL). Theresulting mixture was washed sequentially with saturated aqueous sodiumhydrogenocarbonate solution (50 mL), water (2×50 mL), and saturatedaqueous sodium chloride solution (30 mL). The aqueous layer wasextracted with ethyl acetate (2×100 mL), and the combined organic layerswere dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The resulting yellow residue waspurified by flash column chromatography (eluent: gradient, 10→25%acetone in dichloromethane) to afford the diol (−)-56 (127 mg, 41.6%) asa white solid (Analytically pure samples of polar diol (−)-56 could beobtained by trituration with minimal amount of chloroform). Structuralassignments were made with additional information from gCOSY, HSQC,gHMBC, and NOESY data. ¹H NMR (600 MHz, acetone-ds, 20° C.): δ 9.85(br-s, 1H, N_(1′)H), 8.01 (d, J=8.2, 1H, C_(5′)H), 7.56 (d, J=8.1, 1H,C₈H), 7.49 (d, J=8.1, 1H, C_(8′)H), 7.41 (d, J=7.5, 1H, C₅H), 7.35(app-t, J=7.5, 1H, SO₂Ph-p-H), 7.35 (app-t, J=7.5, 1H, C₇H), 7.24(app-t, J=7.6, 1H, C_(7′)H), 7.20 (app-t, J=7.5, 1H, C₆H), 7.17 (app-t,J=7.5, 1H, C_(6′)H), 7.04 (d, J=7.5, 2H, SO₂Ph-o-H), 6.98 (app-t, J=7.8,2H, SO₂Ph-m-H), 6.80 (d, J=6.2, 1H, C₁₅OH), 6.66 (s, 1H, C₂H), 6.22 (s,1H, C₁₁OH), 5.65 (d, J=2.5, 1H, C_(2′)H), 5.15 (d, J=6.0, 1H, C₁₅H),3.64 (d, J=15.1, 1H, C₁₂H_(a)), 3.01 (d, J=15.1, 1H, C₁₂H_(b)), 2.95 (s,3H, C₁₇H₃). ¹³C NMR (150 MHz, acetone-d₆, 20° C.): δ 168.1 (C₁₃), 165.7(C₁₆), 140.4 (C₉), 139.3 (SO₂Ph-ipso-C), 138.8 (C₄), 138.6 (C_(9′)),133.7 (SO₂Ph-p-C), 129.8 (C₇), 128.9 (SO₂Ph-m-C), 127.5 (SO₂Ph-o-C),126.3 (C₅), 126.2 (C₆), 125.7 (C_(2′)), 125.2 (C_(4′)), 122.9 (C_(7′)),120.4 (C_(6′)), 119.6 (C_(5′)), 118.2 (C₈), 115.7 (C_(3′)), 113.0(C_(8′)), 88.6 (C₁₁), 85.3 (C₂), 83.9 (C₁₅), 55.3 (C₃), 45.1 (C₁₂), 31.8(C₁₇). FTIR (thin film) cm⁻¹: 3392 (br-m), 1700 (s), 1460 (w), 1400 (w),1360 (m), 1169 (m), 1091 (w), 750 (w). HRMS (ESI) (m/z): calc'd forC₂₈H₂₄N₄NaO₆S [M+Na]⁺: 567.1309, found: 567.1315. [α]_(D) ²⁴: −71.4(c=0.114, acetone). m.p.: 212° C. TLC (20% acetone in dichloromethane),Rf: 0.24 (UV, CAM).

C3-(Indol-3′-yl) Epidithiodiketopiperazine 26:

A slow stream of hydrogen sulfide gas was introduced into a solution ofdiol (−)-56 (254 mg, 466 μmol, 1 equiv) in anhydrous nitroethane (20 mL)at 0° C., providing a saturated hydrogen sulfide solution. After 20 min,trifluoroacetic acid (TFA, 15 mL) was added slowly via syringe, and theslow introduction of hydrogen sulfide into the mixture was maintainedfor another 20 min. The reaction mixture was left under an atmosphere ofhydrogen sulfide. The ice-water bath was removed, and the yellowsolution was allowed to warm to 23° C. After 2 h, a slow stream of argongas was introduced into the solution. After 15 min, the reaction mixturewas diluted with ethyl acetate (150 mL) and slowly poured into saturatedaqueous sodium hydrogenocarbonate solution (70 mL) at 23° C. The organiclayer was sequentially washed with water (3×40 mL) and saturated aqueoussodium chloride solution (25 mL), was dried over anhydrous sodiumsulfate, was filtered, and was concentrated under reduced pressure toafford the corresponding bisthiol S13 that was used in the next stepwithout further purification. The orange residue was dissolved in ethylacetate (120 mL). A slow stream of dioxygen gas was introduced into thesolution. After 4 h, the yellow solution was concentrated under reducedpressure. The orange residue was purified by flash column chromatographyon silica gel (eluent: gradient, 5→15% ethyl acetate in dichloromethane)to afford the epidithiodiketopiperazine 26 (205 mg, 76.7%) as a whitesolid. Structural assignments were made with additional information fromgCOSY, HSQC, and gHMBC data. ¹H NMR (600 MHz, acetone-d₆, 20° C.): δ10.05 (br-s, 1H, N_(1′)H), 7.65 (d, J=8.1, 1H, C₈H), 7.55 (d, J=7.5, 1H,C₅H), 7.50 (d, J=8.0, 1H, C_(5′)H), 7.48 (d, J=8.8, 1H, C_(8′)H), 7.46(app-dt, J=1.0, 7.5, 1H, C₇H), 7.39 (t, J=7.4, 1H, SO₂Ph-p-H), 7.30(app-t, J=0.8, 7.5, 1H, C₆H), 7.22 (dd, J=7.2, 8.0, 1H, C_(7′)H), 7.12(app-dd, J=1.0, 8.4, 2H, SO₂Ph-o-H), 7.10 (dd, J=7.3, 7.9, 1H, C_(6′)H),7.00 (dd, J=7.5, 8.2, 2H, SO₂Ph-m-H), 6.63 (s, 1H, C₂H), 5.98 (d, J=2.6,1H, C_(2′)H), 5.80 (s, 1H, C₁₅H), 3.95 (d, J=15.6, 1H, C₁₂H_(a)), 3.17(s, 3H, C₁₇H₃), 2.92 (d, J=15.7, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz,acetone-d₆, 20° C.): δ 165.9 (C₁₃), 161.0 (C₁₆), 141.5 (C₉), 138.7(SO₂Ph-ipso-C), 138.5 (C_(9′)), 138.1 (C₄), 134.0 (SO₂Ph-p-C), 130.1(C₇), 129.0 (SO₂Ph-m-C), 127.7 (SO₂Ph-o-C), 126.6 (C₆), 125.9 (C₅),125.8 (C_(2′)), 125.0 (C_(4′)), 123.0 (C_(7′)), 120.6 (C_(6′)), 119.2(C₈), 119.1 (C_(5′)), 114.1 (C_(3′)), 113.1 (C_(8′)), 85.7 (C₂), 75.5(C₁₁), 69.1 (C₁₅), 56.4 (C₃), 42.6 (C₁₂), 31.8 (C₁₇). FTIR (thin film)cm⁻¹: 3392 (w), 3060 (w), 2990 (w), 1693 (s), 1447 (w), 1358 (m), 1234(w), 1169 (m), 1089 (w), 1052 (w), 964 (w), 736 (m), 587 (m). HRMS (ESI)(m/z): calc'd for C₂₈H₂₃N₄O₄S₃ [M+H]⁺: 575.0876, found 575.0885; calc'dfor C₂₈H₂₂N₄NaO₄S₃ [M+Na]⁺: 597.0695, found 597.0704. TLC (20% ethylacetate in dichloromethane), Rf: 0.62 (UV, CAM).

General Procedure for the Friedel-Crafts Nucleophilic Substitution.

A round-bottom flask was charged with endo-tetracyclic bromide (+)-54 (1equiv, Boyer, N.; Movassaghi, M. Chem. Sci. 2012, 3, 1798),2,6-di-tert-butyl-4-methylpyridine (DTBMP, 2.10 equiv), and thenucleophile (for 68: tetrafluoroborate as nucleophilic fluorine source;for 69: 1-(triisopropylsilyl)-1H-pyrrole; for 70: anisole), and themixture was dried azeotropically (concentration of an anhydrous benzenesolution, 2×10 mL) under reduced pressure and placed under an argonatmosphere. Anhydrous nitroethane (4 mL) was introduced via syringe, andthe mixture was cooled to 0° C. in an ice-water bath. A solution ofsilver(I) tetrafluoroborate (2.30 equiv) in anhydrous nitroethane (1 mL)at 0° C. was introduced via syringe to the solution containing thetetracyclic bromide (+)-54 over 1 min. The reaction flask was covered inaluminum foil. The ice-water bath was removed, and the reaction mixturewas allowed to warm to 23° C. After 1 h, saturated aqueous sodiumchloride solution (10 mL) was introduced, and the resulting biphasicmixture was vigorously stirred for 30 min at 23° C. The reaction mixturewas diluted with ethyl acetate (50 mL), was filtered through a Celitepad, and the solids were washed with ethyl acetate (3×15 mL). Thecombined filtrates were washed with 5% aqueous citric acid solution(2×20 mL), water (3×20 mL), and saturated aqueous sodium chloridesolution (15 mL). The organic layer was dried over anhydrous sodiumsulfate, was filtered, and was concentrated under reduced pressure.

C3-Fluoro Friedel-Crafts adduct 68: ¹H NMR (600 MHz, CDCl₃, 20° C.): δ7.79 (app-dd, J=0.9, 8.2, 2H, SO₂Ph-o-H), 7.56 (d, J=8.2, 1H, C₈H), 7.50(t, J=7.5, 1H, SO₂Ph-p-H), 7.39-7.35 (m, 1H, C₇H), 7.38 (dd, J=7.9, 8.3,2H, SO₂Ph-m-H), 7.34 (d, J=7.7, 1H, C₅H), 7.15 (dd, J=7.5, 7.6, 1H,C₆H), 6.07 (d, J=14.5, 1H, C₂H), 4.53 (dd, J=8.2, 8.4, 1H, C₁₁H), 4.17(d, J=17.6, 1H, C₁₅H_(a)), 3.86 (d, J=17.6, 1H, C₁₅H_(b)), 3.06-2.97 (m,1H, C₁₂H_(a)), 2.93-2.83 (m, 1H, C₁₂H_(b)), 2.90 (s, 3H, C₁₇H₃). ¹⁹F NMR(282.4 MHz, CDCl₃, 20° C.): δ −133.3. MS (ESI) (m/z): [M+H]⁺: 416.22,[M+Na]⁺: 438.25, [2M+H]⁺: 833.73, [2M+Na]⁺: 853.59. TLC (20% acetone indichloromethane), Rf: 0.46 (UV, CAM).

C3-(N-TIPS-Pyrrol-3′-yl) Friedel-Crafts adduct 69: ¹H NMR (600 MHz,CDCl₃, 20° C.): δ 8.03 (app-dd, J=1.0, 7.3, 2H, SO₂Ph-o-H), 7.63 (d,J=7.7, 1H, C₈H), 7.54 (app-dt, J=1.5, 7.5, 1H, SO₂Ph-p-H), 7.43 (app-t,J=7.6, 2H, SO₂Ph-m-H), 7.16-7.11 (m, 1H, C₇H), 7.05-6.99 (m, 2H,C₅H+C₆H), 6.69-6.65 (m, 1H, C_(5′)H), 6.53-5.49 (m, 1H, C_(4′)H), 6.09(s, 1H, C₂H), 5.83-5.79 (m, 1H, C_(2′)H), 4.33 (dd, J=8.2, 8.9, 1H,C₁₁H), 4.10 (d, J=17.8, 1H, C₁₅H_(a)), 3.95 (app-dd, J=2.0, 17.6, 1H,C₁₅H_(b)), 2.99 (s, 3H, C₁₇H₃), 2.84 (dd, J=7.4, 13.3, 1H, C₁₂H_(a)),2.73 (dd, J=10.0, 13.3, 1H, C₁₂H_(b)), 1.40 (app-dsp, J=1.6, 7.5, 3H,SiCH(CH₃)₂), 1.08 (d, J=7.6, 9H, SiCH(CH₃)₂), 1.07 (d, J=6.3, 9H,SiCH(CH₃)₂). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 167.7 (C₁₃), 166.8(C₁₆), 139.5 (C₉), 137.6 (SO₂Ph-ipso-C), 135.9 (C₄), 133.4 (SO₂Ph-p-C),129.0 (SO₂Ph-m-C), 128.9 (C₇), 128.2 (SO₂Ph-o-C), 125.7 (C_(5′)), 125.0(C_(3′)), 124.6 (C₆), 124.0 (C₅), 121.2 (C_(2′)), 115.6 (C₈), 109.4(C_(4′)), 84.8 (C₂), 59.5 (C₁₁), 55.3 (C₃), 54.5 (C₁₅), 39.6 (C₁₂), 33.6(C₁₇), 17.9 (SiCH(CH₃)₂), 11.7 (SiCH(CH₃)₂). MS (ESI) (m/z): [M+H]⁺:619.49, [M+Na]⁺: 641.49, [2M+Na]⁺: 1261.37. TLC (20% acetone indichloromethane), Rf: 0.48 (UV, CAM).

C3-(p-Methoxyphenyl) Friedel-Crafts adduct 70: ¹H NMR (600 MHz, CDCl₃,20° C.): δ 7.60 (app-dd, J=0.7, 8.1, 1H, C₈H), 7.46 (app-dd, J=1.1, 8.4,2H, SO₂Ph-o-H), 7.34 (app-dt, J=1.1, 7.5, 1H, SO₂Ph-p-H), 7.30-7.26 (m,1H, C₇H), 7.14-7.11 (m, 2H, C₅H+C₆H), 7.11 (app-t, J=7.5, 2H,SO₂Ph-m-H), 6.67 (d, J=8.9, 2H, C_(2′)H), 6.63 (d, J=8.9, 1H, C_(3′)H),6.15 (s, 1H, C₂H), 4.42 (dd, J=7.6, 8.2, 1H, C₁₁H), 4.12 (d, J=17.5, 1H,C₁₅H_(a)), 3.84 (d, J=17.5, 1H, C₁₅H_(a)), 3.78 (s, 3H, C_(5′)H₃), 3.10(dd, J=6.8, 14.2, 1H, C₁₂H_(a)), 2.91-2.85 (m, 1H, C₁₂H_(b)), 2.90 (s,3H, C₁₇H₃). MS (ESI) (m/z): [M+Na]⁺: 526.31, [2M+Na]⁺: 1029.94. TLC (20%acetone in dichloromethane), Rf: 0.37 (UV, CAM).

General Procedure for the Regio- and Stereoselective Hydroxylation.

Freshly prepared tetra-n-butylammonium permanganate (4.0 equiv) wasadded as a solid to a solution of the corresponding diketopiperazine(54, 68-70) (1 equiv) in dichloromethane (0.05 M) at 23° C. After 2 h,the dark purple solution was diluted with saturated aqueous sodiumsulfite solution (20 mL) and then with ethyl acetate (120 mL). Theresulting mixture was washed sequentially with saturated aqueous sodiumhydrogenocarbonate solution (20 mL), water (4×20 mL), and saturatedaqueous sodium chloride solution (20 mL). The organic layer was driedover anhydrous sodium sulfate, was filtered, and was concentrated underreduced pressure.

C3-Bromo Epidithiodiketopiperazines 30 and 34:

This compound was prepared in two steps starting from bishemiaminal S14(13.5 mg, 26.6 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine 26. The orangeresidue was purified by flash column chromatography on silica gel(eluent: gradient, 15→40% ethyl acetate in dichloromethane) to affordthe β-epimer of epidithiodiketopiperazine 30 (6.3 mg, 44%) as acolorless oil and its α-epimer 34 (2.1 mg, 15%) as a colorless oil.Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data.

S14:

¹H NMR (600 MHz, MeOD-d₄, 20° C.): δ 7.89 (app-dd, J=0.8, 8.2, 2H,SO₂Ph-o-H), 7.56 (t, J=7.5, 1H, SO₂Ph-p-H), 7.47 (d, J=8.3, 1H, C₈H),7.44 (dd, J=7.5, 8.2 2H, SO₂Ph-m-H), 7.38 (d, J=7.7, 1H, C₅H), 7.33(app-dt, J=1.0, 7.7, 1H, C₇H), 7.16 (app-dt, J=0.6, 7.5, 1H, C₆H), 6.55(s, 1H, C₂H), 4.99 (s, 1H, C₁₅H), 3.71 (d, J=15.4, 1H, C₁₂H_(a)), 3.09(d, J=15.4, 1H, C₁₂H_(b)), 2.86 (s, 3H, C₁₇H₃). MS (ESI) (m/z):[2M+Na]⁺: 1039.24. TLC (20% acetone in dichloromethane), Rf: 0.40 (UV,CAM).

β-Epimer 30:

¹NMR (600 MHz, CDCl₃, 20° C.): 7.82 (d, J=8.0, 2H, SO₂Ph-o-H), 7.60 (d,J=8.2, 1H, C₈H), 7.52 (app-dd, J=7.4, 7.6, 1H, SO₂Ph-p-H), 7.42-7.38 (m,1H, C₇H), 7.40 (app-t, J=7.7, 2H, SO₂Ph-m-H), 7.35 (d, J=7.7, 1H, C₅H),7.25 (app-t, J=7.6, 1H, C₆H), 6.47 (s, 1H, C₂H), 5.22 (s, 1H, C₁₅H),3.82 (d, J=15.4, 1H, C₁₂H_(a)), 3.19 (d, J=15.4, 1H, C₁₂H_(b)), 3.11 (s,3H, C₁₇H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 164.3 (C₁₃), 159.6(C₁₆), 140.2 (C₉), 138.1 (SO₂Ph-ipso-C), 135.1 (C₄), 134.1 (SO₂Ph-p-C),131.5 (C₇), 129.2 (SO₂Ph-m-C), 128.3 (SO₂Ph-o-C), 127.1 (C₆), 124.3(C₅), 118.9 (C₈), 87.4 (C₂), 74.0 (C₁₁), 68.3 (C₁₅), 58.2 (C₃), 46.7(C₁₂), 32.3 (C₁₇). FTIR (thin film) cm⁻¹: 2926 (m), 2857 (w), 1771 (m),1697 (s), 1551 (w), 1449 (m), 1368 (s), 1170 (s), 1090 (w), 1055 (w),756 (s). HRMS (ESI) (m/z): calc'd for C₂₀H₁₆BrN₃NaO₄S₃ [M+Na]⁺:559.9379, found 559.9392. TLC (20% ethyl acetate in dichloromethane),Rf: 0.47 (UV, I₂, CAM). The relative stereochemistry of the epidisulfidebridge of the β-epimer 30 has been confirmed by key NOESY cross-peaks onthe corresponding bis(methylthioether). Assignment is supported by keyNOESY signals (¹H,¹H) in ppm: (1.86, 3.40), (3.40, 7.36), (3.11, 6.68).This derivatized compound was prepared in one step using the methodologydeveloped to access (+)-gliocladin B (Boyer, N.; Movassaghi, M. Chem.Sci. 2012, 3, 1798). {¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.82 (d, J=8.1,2H, SO₂Ph-o-H), 7.59 (d, J=8.2, 1H, C₈H), 7.55 (app-dd, J=7.3, 7.6, 1H,SO₂Ph-p-H), 7.45 (dd, J=7.7, 7.8, 2H, SO₂Ph-m-H), 7.39 (app-t, J=7.9,1H, C₇H), 7.36 (d, J=7.8, 1H, C₅H), 7.19 (app-t, J=7.6, 1H, C₆H), 6.68(s, 1H, C₂H), 4.52 (s, 1H, C₁₅H), 3.40 (d, J=14.5, 1H, C₁₂H_(a)), 3.11(d, J=14.5, 1H, C₁₂H_(b)), 3.06 (s, 3H, C₁₇H₃), 2.27 (s, 3H, C₁₅SCH₃),1.86 (s, 3H, C₁₁SCH₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 164.0 (C₁₃),163.5 (C₁₆), 140.5 (C₉), 138.8 (SO₂Ph-ipso-C), 137.2 (C₄), 133.6(SO₂Ph-p-C), 131.1 (C₇), 129.3 (SO₂Ph-m-C), 127.6 (SO₂Ph-o-C), 125.9(C₆), 123.8 (C₅), 117.9 (C₈), 86.8 (C₂), 69.7 (C₁₁), 67.3 (C₁₅), 58.0(C₃), 49.9 (C₁₂), 32.7 (C₁₇), 17.2 (C₁₅SCH₃), 15.4 (C₁₁SCH₃).}

α-Epimer 34:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.91 (d, J=8.1, 2H, SO₂Ph-o-H),7.53-7.51 (m, 1H, SO₂Ph-p-H), 7.52 (d, J=7.9, 1H, C H), 7.41 (app-t,J=7.7, 2H, SO₂Ph-m-H), 7.38 (d, J=7.9, 1H, C₅H), 7.32 (dd, J=7.6, 8.0,1H, C₇H), 7.17 (app-t, J=7.6, 1H, C₆H), 6.61 (s, 1H, C₂H), 5.16 (s, 1H,C₁₅H), 4.25 (d, J=15.0, 1H, C₁₂H_(a)), 3.09 (d, J=15.0, 1H, C₁₂H_(b)),2.95 (s, 3H, C₁₇H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 164.1 (C₁₃),160.9 (C₁₆), 138.8 (C₉), 138.2 (SO₂Ph-ipso-C), 134.1 (SO₂Ph-p-C), 133.6(C₄), 131.5 (C₇), 129.2 (SO₂Ph-m-C), 128.4 (SO₂Ph-o-C), 126.8 (C₆),125.2 (C₅), 117.7 (C₈), 87.7 (C₂), 73.8 (C₁₁), 68.9 (C₁₅), 58.2 (C₃),45.0 (C₁₂), 31.9 (C₁₇). FTIR (thin film) cm⁻¹: 3296 (w), 3008 (m), 2925(s), 2855 (s), 1771 (m), 1699 (s), 1552 (m), 1463 (s), 1447 (s), 1368(s), 1171 (s), 1091 (s), 1057 (m), 757 (s). HRMS (ESI) (m/z): calc'd forC₂₀H₁₆BrN₃NaO₄S₃ [M+Na]⁺: 559.9379, found 559.9396. TLC (20% ethylacetate in dichloromethane), Rf: 0.56 (UV, I₂, CAM).

C3-Fluoro Epidithiodiketopiperazines 31 and 35:

This compound was prepared in two steps starting from bishemiaminal S16(15.1 mg, 33.7 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine 26. The orangeresidue was purified by flash column chromatography on silica gel(eluent: gradient, 15→40% ethyl acetate in dichloromethane) to affordthe β-epimer of epidithiodiketopiperazine 31 (5.4 mg, 34%) as acolorless oil and its α-epimer 35 (2.1 mg, 13%) as a colorless oil.Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data.

S16:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.68 (d, J=7.6, 2H, SO₂Ph-o-H), 7.56(d, J=8.2, 1H, C₈H), 7.50 (t, J=8.2, 1H, SO₂Ph-p-H), 7.43 (dd, J=7.6,8.2, 1H, C₇H), 7.36 (app-t, J=7.9, 2H, SO₂Ph-m-H), 7.36-7.33 (m, 1H,C₅H), 7.18 (app-t, J=7.5, 1H, C₆H), 6.44 (d, J=13.2, 1H, C₂H), 5.75(br-s, 2H, C₁₁OH+C₁₅OH), 5.13 (s, 1H, C₁₅H), 3.49 (dd, J=8.6, 15.6, 1H,C₁₂H_(a)), 3.02 (s, 3H, C₁₇H₃), 2.97 (dd, J=15.6, 20.8, 1H, C₁₂H_(b)).¹³C NMR (100 MHz, CDCl₃, 20° C.): δ 166.7 (C₁₃), 166.4 (C₁₆), 141.8 (d,J=4.6, C₉), 136.8 (SO₂Ph-ipso-C), 134.0 (SO₂Ph-p-C), 132.2 (d, J=3.2,C₇), 130.6 (d, J=23.5, C₄), 129.2 (SO₂Ph-m-C), 128.0 (SO₂Ph-o-C), 126.8(C₆), 125.5 (C₅), 118.5 (C₈), 101.7 (d, J=202.3, C₃), 88.5 (d, J=4.1,C₁₁), 83.1 (d, J=33.0, C₂), 83.0 (C₁₅), 42.9 (d, J=29.7, C₁₂), 32.6(C₁₇). ¹⁹F NMR (282.4 MHz, CDCl₃, 20° C.): δ −133.2. FTIR (thin film)cm⁻¹: 3365 (br-m), 1695 (br-s), 1447 (m), 1402 (m), 1365 (m), 1342 (m),1173 (m), 1087 (w), 1023 (w), 912 (w), 729 (m), 600 (m). HRMS (ESI)(m/z): calc'd for C₂₀H₁₉FN₃O₆S [M+H]⁺: 448.0973, found 448.0963; calc'dfor C₂₀H₁₈FN₃NaO₆S [M+Na]⁺: 470.0793. found 470.0780. TLC (20% acetonein dichloromethane), Rf: 0.29 (UV, CAM).

β-Epimer 31:

¹H NMR (600 MHz. CDCl₃, 20° C.): δ 7.68 (app-dd, J=1.1, 7.4, 2H,SO₂Ph-o-H), 7.64 (d, J=8.2, 1H, C₈H), 7.51 (t, J=7.5, 1H, SO₂Ph-p-H),7.50 (app-dt, J=1.1, 6.7, 1H, C₇H), 7.38 (dd, J=7.6, 8.1, 2H,SO₂Ph-m-H), 7.40-7.36 (m, 1H, C₆H), 7.28 (d, J=7.6, 1H, C₅H), 6.31 (d,J=11.8, 1H, C₂H), 5.23 (s, 1H, C₁₅H), 3.65 (app-t, J=15.2, 1H,C₁₂H_(a)), 3.13 (s, 3H, C₁₇H₃), 2.89 (app-d, J=15.1, 1H, C₁₂H_(b)). ¹³CNMR (150 MHz, CDCl₃, 20° C.): δ 164.5 (C₁₃), 160.0 (C₁₆), 143.2 (d,J=4.8, C₉), 137.3 (SO₂Ph-ipso-C), 133.8 (SO₂Ph-p-C), 132.8 (d, J=3.4,C₇), 129.9 (d, J=23.3, C₄), 129.1 (SO₂Ph-m-C), 127.9 (SO₂Ph-o-C), 126.8(d, J=2.8, C₆), 124.8 (C₅), 119.4 (d, J=2.2, C₈), 102.3 (d, J=205.5,C₃), 82.7 (d, J=31.8, C₂), 74.4 (d, J=6.2, C₁₁), 68.4 (C₁₅), 39.2 (d,J=32.3, C₁₂), 32.3 (C₁₇). ¹⁹F NMR (282 MHz, CDCl₃, 20° C.): δ −137.7.FTIR (thin film) cm⁻¹: 2999 (w), 2920 (w), 1693 (s), 1447 (w), 1368 (m),1173 (m), 1088 (w), 1040 (w), 914 (w), 719 (w). HRMS (ESI) (m/z): calc'dfor C₂₀H₁₇FN₃O₄S₃ [M+H]⁺: 478.0360, found 478.0375; calc'd forC₂₀H₁₆FN₃NaO₄S₃ [M+Na]⁺: 500.0179, found 500.0198. TLC (20% ethylacetate in dichloromethane), Rf: 0.27 (UV, I₂, CAM). The relativestereochemistry of the epidisulfide bridge of the β-epimer 31 has beenconfirmed by key NOESY cross-peaks on the correspondingbis(methylthioether). Assignment is supported by key NOESY signals (¹H,¹H) in ppm: (1.93, 3.11), (3.11, 7.41), (2.94, 6.45). This derivatizedcompound was prepared in one step using the methodology developed toaccess (+)-gliocladin B (Boyer, N.; Movassaghi, M. Chem. Sci. 2012, 3,1798). {¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.94 (d, J=8.0, 2H,SO₂Ph-o-H), 7.72 (d, J=8.3, 1H, C₈H), 7.56 (app-dd, J=7.4, 7.5, 1H,SO₂Ph-p-H), 7.49-7.45 (m, 1H, C₇H), 7.47 (app-t, J=7.7, 2H, SO₂Ph-m-H),7.41 (d, J=7.7, 1H, C₅H), 7.20 (app-t, J=7.5, 1H, C₆H), 6.45 (d, J=17.5,1H, C₂H), 4.58 (s, 1H, C₁₅H), 3.11 (app-t, J=14.3, 1H, C₁₂H_(a)), 3.09(s, 3H, C₁₇H₃), 2.94 (dd, J=14.3, 20.1, 1H, C₁₂H_(b)), 2.30 (s, 3H,C₁₉H₃), 1.93 (s, 3H, C₂₀H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 164.3(C₁₃), 160.2 (C₁₆), 144.1 (d, J=5.1, C₉), 138.3 (SO₂Ph-ipso-C), 133.6(SO₂Ph-p-C), 132.4 (d, J=3.2, C₇), 129.3 (SO₂Ph-m-C), 128.8 (d, J=23.9,C₄), 127.5 (SO₂Ph-o-C), 125.3 (d, J=2.7, C₆), 124.2 (C₅), 117.1 (d,J=1.8, C₈), 102.8 (d, J=200.8, C₃), 82.0 (d, J=32.5, C₂), 70.6 (d,J=6.5, C₁₁), 67.1 (C₁₅), 45.5 (d, J=31.8, C₁₂), 32.8 (C₁₇), 16.9(C₁₅SCH₃), 15.2 (C₁₁SCH₃). ¹⁹F NMR (282.4 MHz, CDCl₃, 20° C.): δ −135.0.MS (ESI) (m/z): [M+Na]⁺: 530.52, [2M+Na]⁺: 1038.00.)

α-epimer 35:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.74 (d, J=8.5, 2H, SO₂Ph-o-H), 7.59(d, J=8.2, 1H, C₈H), 7.51 (app-dt, J=1.1, 7.6, 1H, SO₂Ph-p-H), 7.43 (dd,J=7.5, 7.6, 1H, C₇H), 7.40 (d, J=7.5, 1H, C₅H), 7.39 (app-t, J=7.6, 2H,SO₂Ph-m-H), 7.20 (dd, J=7.5, 7.6, 1H, C₆H), 6.43 (d, J=11.5, 1H, C₂H),5.21 (s, 1H, C₁₅H), 3.89 (dd, J=5.2, 15.2, 1H, C₁₂H_(a)), 3.01 (s, 3H,C₁₇H₃), 2.85 (app-ddd, J=0.5, 15.9, 16.6, 1H, C₁₂H_(b)). ¹³C NMR (150MHz, CDCl₃, 20° C.): δ 164.2 (C₁₃), 161.6 (C₁₆), 141.9 (d, J=3.7, C₉),137.0 (SO₂Ph-ipso-C), 134.0 (SO₂Ph-p-C), 132.8 (d, J=2.8, C₇), 129.2(SO₂Ph-m-C), 128.6 (d, J=19.5, C₄), 128.0 (SO₂Ph-o-C), 126.8 (C₆), 125.4(C₅), 118.5 (C₈), 101.8 (d, J=170.3, C₃), 83.1 (d, J=26.9, C₂), 74.0 (d,J=4.0, C₁₁), 68.6 (C₁₅), 39.2 (d, J=26.4, C₁₂), 31.9 (C₁₇). ¹⁹F NMR (282MHz, CDCl₃, 20° C.): δ −134.1. FTIR (thin film) cm⁻¹: 3069 (w), 2991(w), 1699 (s), 1448 (w), 1367 (m), 1335 (m), 1173 (m), 1089 (w), 908(w), 730 (m), 720 (m). HRMS (ESI) (m/z): calc'd for C₂₀H₁₇FN₃O₄S₃[M+H]⁺: 478.0360, found 478.0372; calc'd for C₂₀H₁₆FN₃NaO₄S₃ [M+Na]⁺:500.0179, found 500.0199. TLC (20% ethyl acetate in dichloromethane),Rf: 0.16 (UV, I₂, CAM).

C3-(Pyrrol-3′-yl) Epidithiodiketopiperazine 32:

This compound was prepared in two steps starting from bishemiaminal S18(308 mg, 473 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine 26. The orangeresidue was purified by flash column chromatography on silica gel(eluent: gradient, 10→40% ethyl acetate in dichloromethane) to affordthe epidithiodiketopiperazine 32 (128 mg, 51.5%) as a pale yellow solid.Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data. The relative stereochemistry of the epidisulfidebridge 32 has been confirmed by key NOESY cross-peaks on thecorresponding bis(methylthioether). Assignment is supported by key NOESYsignals (¹H,¹H) in ppm: (1.89, 3.06), (2.91, 5.86-5.82), (2.91,6.07-6.04), (2.91, 6.47). This derivatized compound was prepared in onestep using the methodology developed to access (+)-gliocladin B (Boyer,N.; Movassaghi, M. Chem. Sci. 2012, 3, 1798). {¹H NMR (600 MHz, CDCl₃,20° C.): δ 8.07 (br-s, 1H, N_(1′)H), 7.84 (d, J=7.5, 2H, SO₂Ph-o-H),7.50 (d, J=8.2, 1H, C₈H), 7.47 (t, J=7.5, 1H, SO₂Ph-p-H), 7.35 (app-t,J=7.9, 2H, SO₂Ph-m-H), 7.28 (app-dt, J=1.1, 7.8, 1H, C₇H), 7.19 (d,J=7.4, 1H, C₅H), 7.09 (dd, J=7.4, 7.5, 1H, C₆H), 6.65-6.62 (m, 1H,C_(5′)H), 6.47 (s, 1H, C₂H), 6.07-6.04 (m, 1H, C_(2′)H), 5.86-5.82 (m,1H, C_(4′)H), 4.50 (s, 1H, C₁₅H), 3.06 (d, J=14.4, 1H, C₁₂H_(a)), 3.06(s, 3H, C₁₈H₃), 2.91 (d, J=14.4, 1H, C₁₂H_(b)), 2.23 (s, 3H, C₁₅SCH₃),1.89 (s, 3H, C₁₁SCH₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 165.3 (C₁₃),162.6 (C₁₆), 142.1 (C₉), 140.0 (SO₂Ph-ipso-C), 137.5 (C₄), 132.8(SO₂Ph-p-C), 128.9 (SO₂Ph-m-C), 128.8 (C₇), 127.1 (SO₂Ph-o-C), 125.7(C_(3′)), 124.9 (C₆), 123.5 (C₅), 119.3 (C_(5′)), 117.0 (C₈), 115.6(C_(2′)), 106.3 (C_(4′)), 86.0 (C₂), 69.7 (C₁₁), 67.7 (C₁₅), 53.1 (C₃),45.7 (C₁₂), 32.5 (C₁₇), 17.3 (C₁₅SCH₃), 15.5 (C₁₁SCH₃).} ¹H NMR (600MHz, CDCl₃, 20° C.): δ 7.86 (br-s, 1H, N_(1′)H), 7.57 (d, J=8.1, 1H,C₈H), 7.49 (d, J=8.4, 2H, SO₂Ph-o-H), 7.36 (app-dt, J=1.1, 7.6, 1H,SO₂Ph-p-H), 7.35 (app-t, J=8.2, 1H, C₇H), 7.23 (d, J=7.5, 1H, C₅H), 7.19(dd, J=7.4, 7.5, 1H, C₆H), 7.15 (app-dt, J=0.9, 7.4, 2H, SO₂Ph-m-H),6.72-6.69 (m, 1H, C_(5′)H), 6.28 (s, 1H, C₂H), 6.03-5.99 (m, 1H,C_(4′)H), 5.58-5.54 (m, 1H, C_(2′)H), 5.22 (s, 1H, C₁₅H), 3.60 (d,J=15.5, 1H, C₁₂H_(a)), 3.13 (s, 3H, C₁₇H₃), 2.82 (d, J=15.5, 1H,C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 165.4 (C₃), 160.3 (C₁₆),140.9 (C₉), 138.6 (SO₂Ph-ipso-C), 137.2 (C₄), 132.9 (SO₂Ph-p-C), 129.5(C₇), 128.5 (SO₂Ph-m-C), 127.6 (SO₂Ph-o-C), 126.0 (C₆), 124.5 (C₅),123.5 (C_(3′)), 119.6 (C_(5′)), 118.5 (C₈), 117.1 (C_(2′)), 106.4(C_(4′)), 87.1 (C₂), 74.4 (C₁₁), 68.4 (C₁₅), 55.4 (C₃), 44.2 (C₁₂), 32.2(C₁₇). FTIR (thin film) cm⁻¹: 3391 (w), 2925 (w), 1699 (s), 1458 (m),1360 (m), 1169 (m), 1090 (w), 749 (m). HRMS (ESI) (m/z): calc'd forC₂₄H₂₀N₄NaO₄S₃ [M+Na]⁺: 547.0539, found 547.0560. TLC (20% ethyl acetatein dichloromethane), Rf: 0.33 (UV, I₂, CAM).

S18:

¹H NMR (600 MHz, acetone-d₆, 20° C.): δ 7.75 (app-dd, J=0.8, 7.5, 2H,SO₂Ph-o-H), 7.54 (app-dt, J=0.9, 7.5, 1H, SO₂Ph-p-H), 7.37 (app-dt,J=0.7, 7.6, 2H, SO₂Ph-m-H), 7.30 (d, J=7.6, 1H, C₈H), 7.26 (app-dd,J=0.4, 7.7, 1H, C₅H), 7.22 (app-dt, J=1.1, 7.6, 1H, C₆H), 7.12 (app-dt,J=1.1, 7.4, 1H, C₇H), 6.72-6.69 (m, 1H, C_(5′)H), 6.66-6.63 (m, 1H,C_(4′)H), 6.39 (s, 1H, C₂H), 6.25 (br-s, 1H, C₁₁OH), 6.09 (br-s, 1H,C₁₅OH), 5.71-5.68 (m, 1H, C_(2′)H), 5.05 (s, 1H, C₁₅H), 3.35 (d, J=14.7,1H, C₁₂H_(a)), 2.92 (s, 3H, C₁₇H₃), 2.85 (d, J=14.7, 1H, C₁₂H_(b)), 1.46(sp, J=7.5, 3H, SiCH(CH₃)₂), 1.08 (d, J=7.5, 18H, SiCH(CH₃)₂). MS (ESI)(m/z): [M+Na]⁺: 547.0539. TLC (20% acetone in dichloromethane), Rf: 0.44(UV, I₂, CAM).

C3-(p-Methoxyphenyl) epidithiodiketopiperazine 33:

This compound was prepared in two steps starting from bishemiaminal S20(380 mg, 709 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine 26. The orangeresidue was purified by flash column chromatography on silica gel(eluent: gradient, 5→25% ethyl acetate in dichloromethane) to afford theepidithiodiketopiperazine 33 (321 mg, 80.0%) as a pale yellow solid.Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data. The relative stereochemistry of the epidisulfidebridge 33 has been confirmed by key NOESY cross-peaks on thecorresponding bis(methylthioether). Assignment is supported by key NOESYsignals (¹H,¹H) in ppm: (1.89, 3.13), (3.13, 7.13-7.07), (2.98, 6.89),(2.98, 6.47). This derivatized compound was prepared in one step usingthe methodology developed to access (+)-gliocladin B (Boyer, N.;Movassaghi, M. Chem. Sci. 2012, 3, 1798). {¹H NMR (600 MHz, CDCl₃, 20°C.): δ 7.85 (app-dd, J=0.7, 7.7, 2H, SO₂Ph-o-H), 7.54 (d, J=8.1, 1H,C₈H), 7.48 (t, J=7.3, 1H, SO₂Ph-p-H), 7.35 (app-t, J=7.8, 2H,SO₂Ph-m-H), 7.30 (app-dt, J=1.4, 7.5, 1H, C₇H), 7.13-7.07 (m, 2H,C₅H+C₆H), 6.89 (d, J=8.8, 2H, C_(2′)H), 6.70 (d, J=8.8, 2H, C_(3′)H),6.64 (s, 1H, C₂H), 4.48 (s, 1H, C₁₅H), 3.75 (s, 3H, C_(5′)H₃), 3.13 (d,J=14.3, 1H, C₁₂H_(a)), 3.06 (s, 3H, C₁₇H₃), 2.98 (d, J=14.3, 1H,C₁₂H_(b)), 2.20 (s, 3H, C₁₅SCH₃), 1.89 (s, 3H, C₁₁SCH₃). ¹³C NMR (150MHz. CDCl₃, 20° C.): δ 165.1 (C₁₃), 162.3 (C₁₆), 158.8 (C_(4′)), 142.3(C₉), 140.1 (SO₂Ph-ipso-C), 136.7 (C₄), 134.2 (C_(1′)), 132.9(SO₂Ph-p-C), 129.1 (SO₂Ph-m-C), 127.1 (C_(2′)), 127.1 (C₇), 127.0(SO₂Ph-o-C), 124.9 (C₆), 123.8 (C₅), 117.1 (C₈), 114.4 (C_(3′)), 85.8(C₂), 69.8 (C₁₁), 67.6 (C₁₅), 57.0 (C₃), 55.5 (C_(5′)), 45.7 (C₁₂), 32.5(C₁₇), 17.2 (C₁₅SCH₃), 15.5 (C₁₁SCH₃).} ¹H NMR (600 MHz, CDCl₃, 20° C.):δ 7.64 (d, J=8.0, 1H, C₈H), 7.40 (app-dt, J=1.4, 7.0, 1H, C₇H), 7.33 (d,J=8.0, 2H, SO₂Ph-o-H), 7.29 (t, J=7.5, 1H, SO₂Ph-p-H), 7.28-7.23 (m, 2H,C₅H+C₆H), 7.02 (dd, J=7.6, 7.8, 2H, SO₂Ph-m-H), 6.76 (d, J=8.7, 2H,C_(2′)H), 6.62 (d, J=8.7, 1H, C_(3′)H), 6.39 (s, 1H, C₂H), 5.28 (s, 1H,C₁₅H), 3.78 (s, 3H, C_(5′)H₃), 3.63 (d, J=15.6, 1H, C₁₂H_(a)), 3.13 (s,3H, C₁₇H₃), 2.87 (d, J=15.6, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20°C.): δ 165.2 (C₁₃), 160.2 (C₁₆), 158.9 (C_(4′)), 141.3 (C₉), 138.3(SO₂Ph-ipso-C), 135.8 (C₄), 133.1 (SO₂Ph-p-C), 131.2 (C_(1′)), 129.9(C₇), 128.6 (SO₂Ph-m-C), 128.0 (C_(2′)), 127.3 (SO₂Ph-o-C), 126.2 (C₆),125.8 (C₅), 119.1 (C₅), 114.5 (C_(3′)), 87.8 (C₂), 74.6 (C₁₁), 68.4(C₁₅), 59.5 (C₃), 55.5 (C_(5′)), 45.6 (C₁₂), 32.2 (C₁₇). FTIR (thinfilm) cm⁻¹: 3065 (w), 3006 (w), 2931 (w), 2839 (w), 1698 (s), 1512 (m),1459 (m), 1363 (m), 1255 (m), 1170 (m), 1035 (w), 755 (m). HRMS (ESI)(m/z): calc'd for C₂₇H₂₃N₃NaO₄S₃ [M+Na]⁺: 588.0692, found 588.0694. TLC(20% ethyl acetate in dichloromethane), Rf: 0.42 (UV, I₂, CAM).

S20:

¹H NMR (600 MHz, DMSO-d₆, 20° C.): δ 7.44 (t, J=7.5, 1H, SO₂Ph-p-H),7.38 (d, J=7.8, 1H, C₈H), 7.34 (app-t, J=8.8, 1H, C₇H), 7.26 (d, J=7.4,2H, SO₂Ph-o-H), 7.21 (app-dt, J=0.6, 7.3, 1H, C₆H), 7.14 (dd, J=7.6,8.1, 2H, SO₂Ph-m-H), 7.01 (d, J=7.5, 1H, C₅H), 6.76 (d, J=8.8, 2H,C_(2′)H), 6.66 (d, J=8.8, 1H, C_(3′)H), 6.22 (s, 1H, C₂H), 5.00 (d,J=7.4, 1H, C₁₅H), 3.74 (s, 3H, C_(5′)H3), 3.19 (d, J=15.0, 1H,C₁₂H_(a)), 2.77 (s, 3H, C₁₇H₃), 2.67 (d, J=15.0, 1H, C₁₂H_(b)). ¹³C NMR(100 MHz, DMSO-d₆, 20° C.): δ 166.6 (C₁₃), 165.8 (C₁₆), 158.0 (C_(4′)),139.4 (C₉), 138.0 (SO₂Ph-ipso-C), 137.8 (C₄), 133.4 (C_(1′)), 133.2(SO₂Ph-p-C), 128.9 (C₇), 128.7 (SO₂Ph-m-C), 128.0 (C_(2′)), 126.7(SO₂Ph-o-C), 126.7 (C₅), 125.7 (C₆), 117.0 (C₈), 114.0 (C_(3′)), 87.3(C₂), 86.0 (C₁₁), 80.9 (C₁₅), 57.4 (C₃), 55.1 (C_(5′)), 49.7 (C₁₂), 30.5(C₁₇). MS (ESI) (m/z): [M+H]⁺: 537.39, [M+Na]⁺: 558.43, [2M+Na]⁺:1094.13. TLC (20% acetone in dichloromethane), Rf: 0.50 (UV, CAM).

Hexacyclic triphenylmethanedisulfide (+)-71:

(Boyer, N.; Movassaghi, M. Chem. Sci. 2012, 3, 1798) Anhydrous hydrazine(150 μL, 4.77 mmol, 11.1 equiv) was added via syringe to a solution ofaminothioisobutyrate (+)-51 (240 mg, 428 μmol, 1 equiv, Boyer, N.;Movassaghi, M. Chem. Sci. 2012, 3, 1798) in anhydrous tetrahydrofuran(50 mL) at 0° C. After 1 h, the reaction mixture was dilutedsequentially with saturated aqueous ammonium chloride solution (20 mL)and ethyl acetate (120 mL). The organic layer was sequentially washedwith saturated aqueous ammonium chloride solution (50 mL), water (2×50mL), and saturated aqueous sodium chloride solution (30 mL). The aqueouslayer was extracted with ethyl acetate (2×50 mL). The combined organiclayers were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure to afford the hexacyclic aminothiolthat was used in the next step without further purification. Thehexacyclic aminothiol can be purified by flash column chromatography onsilica gel (eluent: gradient, 1→3% methanol in dichloromethane). ¹H NMR(600 MHz, CDCl₃, 20° C.): δ 8.16 (br-s, 1H, N_(1′)H), 7.40 (d, J=8.1,1H, C_(5′)H), 7.31 (d, J=8.2, 1H, C_(8′)H), 7.23 (d, J=7.4, 1H, C₅H),7.19 (app-dt, J=1.0, 7.7, 1H, C₇H), 7.17 (app-t, J=7.4, 1H, C_(7′)H),7.02 (app-t, J=7.6, 1H, C_(6′)H), 6.89 (d, J=2.6, 1H, C_(2′)H), 6.85(app-t, J=7.0, 1H, C₆H), 6.77 (d, J=7.8, 1H, C₇H), 5.92 (s, 1H, C₂H),5.36 (s, 1H, C₁₅H), 5.20 (br-s, 1H, N₁H), 3.76 (d, J=14.3, 1H,C₁₂H_(a)), 3.75 (br-s, 1H, C₁₅OH), 3.30 (d, J=14.3, 1H, C₁₂H_(b)), 3.09(s, 3H, C₁₄H₃), 2.57 (br-s, 1H, C₁₁SH). ¹³C NMR (150 MHz, CDCl₃, 20°C.): δ 166.6 (C₁₃), 166.6 (C₁₆), 148.2 (C₉), 137.4 (C_(9′)), 131.8 (C₄),129.4 (C₇), 125.0 (C_(4′)), 125.0 (C₅), 122.7 (C_(7′)), 122.2 (C_(2′)),120.4 (C₆), 120.2 (C_(6′)), 119.7 (C_(5′)), 117.3 (C_(3′)), 111.8(C_(8′)), 110.4 (C₈), 82.5 (C₂), 77.2 (C₁₅), 69.0 (C₁₁), 54.2 (C₃), 50.9(C₁₂), 29.3 (C₁₈). TLC (5% methanol in dichloromethane), Rf: 0.27 (UV,CAM).

Triethylamine (600 μL, 4.27 mmol, 10.0 equiv) and solidtriphenylmethanesulfenyl chloride (665 mg, 2.14 mmol, 5.00 equiv) weresequentially added to a solution of hexacyclic aminothiol in anhydroustetrahydrofuran (60 mL) at 0° C. under an argon atmosphere. After 90min, the solution was partitioned between saturated aqueous ammoniumchloride (50 mL) and ethyl acetate (130 mL). The aqueous layer wasextracted with diethyl ether (2×50 mL), and the combined organic layerswere washed sequentially with water (2×50 mL) and saturated aqueoussodium chloride solution (30 mL), were dried over anhydrous sodiumsulfate, were filtered, and were concentrated under reduced pressure.The residue was purified by flash column chromatography on silica gel(eluent: gradient, 10-30% ethyl acetate in dichloromethane) to affordtriphenylmethanedisulfide (+)-71 (242 mg, 81.4%) as a white solid. Thissequence can also be combined as a sequential single-flask two-stepprocess to afford (+)-71 in 74% yield. Structural assignments were madewith additional information from gCOSY, HSQC, and gHMBC data. ¹H NMR(600 MHz, acetonitrile-d₃, 20° C.): δ 9.16 (br-s, 1H, N_(1′)H), 7.37 (d,J=7.4, 1H, C_(8′)H), 7.36 (d, J=7.6, 1H, C_(5′)H), 7.34-7.30 (m, 6H,C(Ph-o-H)₃), 7.34-7.30 (m, 3H, C(Ph-p-H)₃), 7.18-7.15 (m, 6H,C(Ph-m-H)₃), 7.15-7.11 (m, 1H, C₇H), 7.10 (app-dt, J=0.8, 7.6, 1H,C_(7′)H), 6.97 (d, J=2.7, 1H, C_(2′)H), 6.96 (app-t, J=8.0, 1H,C_(6′)H), 6.68 (d, J=7.9, 1H, C₈H), 6.64-6.60 (m, 1H, C₅H), 6.64-6.60(m, 1H, C₆H), 5.75 (d, J=1.0, 1H, C₂H), 5.60 (br-s, 1H, N₁H), 5.11 (s,1H, C₁₅H), 4.59 (br-s, 1H, C₁₅OH), 3.32 (d, J=14.5, 1H, C₁₂H_(a)), 2.89(s, 3H, C₁₇H₃), 2.70 (d, J=14.5, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz,acetonitrile-d₃, 20° C.): δ 166.9 (C₁₃), 164.1 (C₁₆), 149.2 (C₉), 145.0(C(Ph-ipso-C)₃), 138.1 (C_(9′)), 133.3 (C₄), 131.2 (C(Ph-m-C)₃), 129.4(C₇), 128.8 (C(Ph-o-C)₃), 128.4 (C(Ph-p-C)₃), 125.8 (C_(4′)), 125.4(C₅), 122.8 (C_(7′)), 122.7 (C_(2′)), 120.3 (C₆), 120.3 (C_(5′)), 120.1(C_(6′)), 118.6 (C_(3′)), 112.7 (C_(8′)), 110.6 (C₈), 83.1 (C₂), 78.4(CPh₃), 78.4 (C₁₅), 73.1 (C₁₁), 54.3 (C₃), 49.4 (C₁₂), 29.1 (C₁₈). FTIR(thin film) cm⁻¹: 3345 (br-m), 3056 (w), 2926 (w), 1674 (s), 1483 (m),1459 (m), 1442 (m), 1388 (m), 745 (s), 700 (s). HRMS (ESI) (m/z): calc'dfor C₄₁H₃₅N₄O₃S₂ [M+H]⁺: 695.2145, found: 695.2147. [α]_(D) ²⁴: +165.2(c=0.12, CHCl₃). TLC (5% methanol in dichloromethane), Rf: 0.44 (UV,CAM). Triphenylmethanedisulfide (+)-71 has also been characterized byNMR in CDCl₃: ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.00 (br-s, 1H,N_(1′)H), 7.31 (d, J=7.8, 1H, C_(5′)H), 7.30 (d, J=7.8, 1H, C_(8′)H),7.29-7.26 (m, 6H, C(Ph-o-H)₃), 7.29-7.26 (m, 3H, C(Ph-p-H)₃), 7.20-7.17(m, 6H, C(Ph-m-H)₃), 7.16 (app-t, J=7.7, 1H, C₇H), 7.15 (app-t, J=8.1,1H, C_(7′)H), 7.02 (app-t, J=7.5, 1H, C_(6′)H), 6.83 (d, J=2.5, 1H,C_(2′)H), 6.74-6.68 (m, 1H, C₅H), 6.74-6.68 (m, 1H, C₆H), 6.74-6.68 (m,1H, C₈H), 5.82 (s, 1H, C₂H), 5.24 (d, J=3.6, 1H, C₁₅H), 4.99 (br-s, 1H,N₁H), 4.07 (d, J=3.6, 1H, C₁₅OH), 3.43 (d, J=14.7, 1H, C₁₂H_(a)), 3.00(s, 3H, C₁₇H₃), 2.57 (d, J=14.7, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃,20° C.): δ 167.3 (C₁₆), 163.7 (C₁₃), 147.6 (C₉), 143.9 (C(Ph-ipso-C)₃),137.3 (C_(9′)), 131.6 (C₄), 130.8 (C(Ph-m-C)₃), 129.5 (C₇), 127.9(C(Ph-o-C)₃), 127.5 (C(Ph-p-C)₃), 125.2 (C₅), 125.1 (C_(4′)), 122.6(C_(7′)), 122.0 (C_(2′)), 120.2 (C_(6′)), 120.0 (C_(5′)), 119.9 (C₆),117.5 (C_(3′)), 111.6 (C_(8′)), 110.1 (C₈), 82.6 (C₂), 77.6 (CPh₃), 72.9(C₁₅), 69.7 (C₁₁), 54.0 (C₃), 48.0 (C₁₂), 29.4 (C₁₈).

(+)-12-Deoxybionectin A (10):

(Zheng, C.-J.; Kim, C.-J.; Bae, K. S.; Kim, Y.-H.; Kim, W.-G. J. Nat.Prod. 2006, 69, 1816; Boyer, N.; Movassaghi, M. Chem. Sci. 2012, 3,1798) Hafnium(IV) trifluoromethanesulfonate hydrate (800 mg) was addedas a solid to a colorless solution of hexacyclictriphenylmethanedisulfide (+)-71 (100 mg, 144 μmol, 1 equiv) inanhydrous acetonitrile (40 mL) at 23° C. A bright yellow coloration wasobserved immediately after the addition. The suspension was stirred at23° C. under an argon atmosphere. After 15 min, the reaction mixture waspartitioned between saturated aqueous sodium bicarbonate (60 mL) andethyl acetate (100 mL). The aqueous layer was extracted with ethylacetate (2×50 mL). The combined organic layers were washed sequentiallywith water (3×50 mL) and saturated aqueous sodium chloride solution (30mL), were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 1→6% acetone indichloromethane) to afford (+)-12-deoxybionectin A (10) (50.2 mg, 80.3%)as a colorless oil. Structural assignments were made with additionalinformation from gCOSY, HSQC, and gHMBC data. ¹H NMR (600 MHz, CDCl₃,20° C.): δ 8.07 (br-s, 1H, N_(1′)H), 7.48 (d, J=8.0, 1H, C_(5′)H), 7.37(d, J=8.2, 1H, C_(8′)H), 7.25 (d, J=8.3, 1H, C₅H), 7.20 (app-dt, J=0.7,7.7, 1H, C₇H), 7.20 (app-dt, J=0.7, 7.7, 1H, C_(7′)H), 7.09 (app-t,J=7.6, 1H, C_(6′)H), 6.95 (d, J=2.5, 1H, C_(2′)H), 6.88 (app-t, J=7.4,1H, C₆H), 6.76 (d, J=7.9, 1H, C₈H), 5.95 (s, 1H, C₂H), 5.30 (br-s, 1H,N₁H), 5.21 (s, 1H, C₁₅H), 4.10 (d, J=15.4, 1H, C₁₂H_(a)), 3.15 (s, 3H,C₁₇H₃), 2.95 (d, J=15.4, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20°C.): δ 165.8 (C₁₃), 162.2 (C₁₆), 148.2 (C₉), 137.5 (C_(9′)), 132.0 (C₄),129.4 (C₇), 125.1 (C_(4′)), 124.3 (C₅), 122.9 (C_(7′)), 122.9 (C_(2′)),120.4 (C_(6′)), 120.1 (C₆), 119.6 (C_(5′)), 116.7 (C_(3′)), 111.9(C_(8′)), 110.4 (C₈), 83.0 (C₂), 74.8 (C₁₁), 68.4 (C₁₅), 56.1 (C₃), 43.6(C₁₂), 32.2 (C₁₇). FTIR (thin film) cm⁻¹: 3358 (br-w), 3006 (w), 2926(w), 1684 (s), 1609 (w), 1460 (w), 1383 (w), 1232 (m), 748 (m). HRMS(ESI) (m/z): calc'd for C₂₂H₁₉N₄O₂S₂ [M+H]⁺: 435.0944, found: 435.0943.[α]_(D) ²⁴: +387.3 (c=0.10, CHCl₃). TLC (10% acetone indichloromethane), Rf: 0.54 (UV, CAM). The relative stereochemistry ofthe epidisulfide bridge 10 has been confirmed by key NOESY signals(¹H,¹H) in ppm: (1.99, 3.31), (3.31, 7.16), (3.20, 6.06) on thecorresponding bis(methylthioether)—i.e., (+)-gliocladin B (7, see Boyer,N.; Movassaghi, M. Chem. Sci. 2012, 3, 1798).

C3-(Indol-3′-yl) Epitrithiodiketopiperazine 29:

This compound was prepared in two steps starting fromaminothioisobutyrate (+)-51 (26.5 mg, 47.3 μmol, Boyer, N.; Movassaghi,M. Chem. Sci. 2012, 3, 1798) using the methodology developed to accessthe corresponding C3-(indol-3′-yl) epidithiodiketopiperazine(+)-12-deoxybionectin A (10). The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 2→10% acetone indichloromethane) to afford epitrithiodiketopiperazine 29 (10.3 mg,46.7%) as a colorless oil. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. Without theintention to be limited by theory, Applicant notes that in someembodiments, upon concentration or in concentrated solution, theepitrithiodiketopiperazine 29 tends to degrade, thus rendering itsisolation and characterization particularly arduous. ¹H NMR (600 MHz,CDCl₃, 20° C.): Major conformer: δ 8.10 (br-s, 1H, N_(1′)H), 7.46 (d,J=8.1, 1H, C_(5′)H), 7.35 (d, J=8.0, 1H, C_(8′)H), 7.21 (app-dt, J=0.7,6.9, 1H, C₇H), 7.18 (app-t, J=7.6, 1H, C_(7′)H), 7.14 (d, J=7.3, 1H,C₅H), 7.06 (app-t, J=7.5, 1H, C_(6′)H), 6.92 (d, J=2.4, 1H, C_(2′)H),6.81 (d, J=8.3, 1H, C₈H), 6.80 (app-t, J=7.5, 1H, C₆H), 5.85 (s, 1H,C₂H), 4.87 (s, 1H, C₁₅H), 3.80 (d, J=14.6, 1H, C₁₂H_(a)), 3.20 (s, 3H,C₁₇H₃), 3.16 (d, J=14.6, 1H, C₁₂H_(b)). The resonance for N₁H was notobserved. Minor conformer: δ 8.11 (br-s, 1H, N_(1′)H), 7.54 (d, J=8.1,1H, C_(5′)H), 7.36 (d, J=7.9, 1H, C_(8′)H), 7.22-7.18 (m, 1H, C_(7′)H),7.12 (d, J=7.4, 1H, C₅H), 7.11 (dd, J=7.6, 7.7, 1H, C₇H), 7.09 (app-t,J=7.6, 1H, C_(6′)H), 6.94 (d, J=2.4, 1H, C_(2′)H), 6.78 (app-t, J=7.5,1H, C₆H), 6.71 (d, J=7.7, 1H, C₈H), 6.19 (s, 1H, C₂H), 5.21 (s, 1H,C₁₅H), 3.70 (d, J=14.7, 1H, C₁₂H_(a)), 3.09 (d, J=14.7, 1H, C₁₂H_(b)),3.02 (s, 3H, C₁₇H₃). The resonance for N₁H was not observed. ¹³C NMR(150 MHz, CDCl₃, 20° C.): Major conformer: δ 168.9 (C₁₃), 164.5 (C₁₆),149.6 (C₉), 137.3 (C_(9′)), 130.8 (C₄), 129.9 (C₇), 125.0 (C_(4′)),124.8 (C₅), 122.8 (C_(7′)), 122.5 (C_(2′)), 120.3 (C_(6′)), 120.0 (C₆),119.7 (C_(5′)), 116.5 (C_(3′)), 111.8 (C_(8′)), 110.6 (C₈), 82.1 (C₂),79.3 (C₁₁), 67.2 (C₁₅), 54.2 (C₃), 49.2 (C₁₂), 31.2 (C₁₇). Minorconformer: δ 167.4 (C₁₃), 163.2 (C₁₆), 148.2 (C₉), 137.4 (C_(9′)), 131.4(C₄), 129.2 (C₇), 125.1 (C_(4′)), 124.3 (C₅), 122.8 (C_(7′)), 122.5(C_(2′)), 120.3 (C_(6′)), 120.2 (C₆), 119.7 (C_(5′)), 116.7 (C_(3′)),111.9 (C_(8′)), 109.8 (C₈), 83.7 (C₂), 74.8 (C₁₁), 71.2 (C₁₅), 54.3(C₃), 46.8 (C₁₂), 32.5 (C₁₇). FTIR (thin film) cm⁻¹: 3397 (br-m), 3061(w), 2922 (w), 2852 (w), 1693 (s), 1458 (m), 1382 (m), 1265 (w), 1170(m), 1092 (w), 1026 (w), 737 (m). HRMS (ESI) (m/z): calc'd forC₂₂H₁₉N₄O₂S₄ [M+H]⁺: 467.0665, found: 467.0669. TLC (10% acetone indichloromethane), Rf: 0.61 (UV, I₂, CAM).

C3-(Indol-3′-yl) C11-Thiohemiaminal 48:

A slow stream of hydrogen sulfide gas was introduced into a solution ofdiol (−)-56 (185 mg, 340 μmol, 1 equiv) in anhydrous dichloromethane (30mL) at 0° C., providing a saturated hydrogen sulfide solution. After 20min, trifluoroacetic acid (6 mL) was added via syringe over 10 min, andthe slow introduction of hydrogen sulfide into the mixture wasmaintained for another 10 min. The reaction mixture was left under anatmosphere of hydrogen sulfide for an additional 2 h at 0° C. A slowstream of argon gas was introduced into the solution. After 15 min, thereaction mixture was diluted with ethyl acetate (150 mL) and slowlypoured into saturated aqueous sodium hydrogenocarbonate solution (50mL). The organic layer was sequentially washed with water (3×40 mL) andsaturated aqueous sodium chloride solution (40 mL). The organic layerwas dried over anhydrous sodium sulfate, was filtered, and wasconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 5→25% acetone indichloromethane) to afford thiohemiaminal 48 (171 mg, 89.8%) as anorange solid. Structural assignments were made with additionalinformation from gCOSY, HSQC, and gHMBC data. ¹H NMR (600 MHz, CDCl₃,20° C.): δ 7.89 (br-s, 1H, N_(1′)H), 7.87 (d, J=8.2, 1H, C₈H), 7.45 (d,J=7.7, 2H, SO₂Ph-o-H), 7.44-7.39 (m, 1H, C₇H), 7.34 (t, J=7.4, 1H,SO₂Ph-p-H), 7.31 (d, J=8.2, 1H, C_(8′)H), 7.21-7.18 (m, 2H, C₅H+C₆H),7.17 (dd, J=7.4, 7.8, 1H, C_(7′)H), 7.03 (app-t, J=7.8, 2H, SO₂Ph-m-H),6.92 (dd, J=7.5, 7.6, 1H, C_(6′)H), 6.73 (d, J=8.0, 1H, C_(5′)H), 6.61(s, 1H, C₂H), 6.22 (d, J=2.3, 1H, C_(2′)H), 5.42 (s, 1H, C₁₅H), 4.53(br-s, 1H, C₁₅OH), 3.82 (d, J=14.9, 1H, C₁₂H_(a)), 3.11 (s, 3H, C₁₇H₃),2.99 (d, J=14.9, 1H, C₁₂H_(b)), 2.61 (s, 1H, C₁₁SH). ¹³C NMR (150 MHz,CDCl₃, 20° C.): δ 166.2 (C₁₃), 165.8 (C₁₆), 140.9 (C₉), 137.3(SO₂Ph-ipso-C), 136.8 (C_(9′)), 135.9 (C₄), 133.3 (SO₂Ph-p-C), 129.4(C₇), 128.6 (SO₂Ph-m-C), 127.5 (SO₂Ph-o-C), 126.0 (C₆), 125.0 (C₅),124.1 (C_(4′)), 123.9 (C_(2′)), 122.9 (C_(7′)), 120.5 (C_(6′)), 118.7(C_(5′)), 118.4 (C₈), 114.2 (C_(3′)), 111.9 (C_(8′)), 84.5 (C₂), 77.3(C₁₅), 69.5 (C₁₁), 53.8 (C₃), 51.8 (C₁₂), 29.3 (C₇). FTIR (thin film)cm⁻¹: 3394 (br-w), 2926 (w), 2547 (w), 1700 (s), 1662 (s), 1457 (m),1359 (m), 1168 (s), 1090 (m), 1024 (w), 734 (m). HRMS (ESI) (m/z):calc'd for C₂₈H₂₄N₄NaO₅S₂ [M+Na]⁺: 583.1080, found: 583.1095. TLC (20%ethyl acetate in dichloromethane), Rf: 0.09 (UV, CAM).

C3-(Indo-3′-yl) Epitrithiodiketopiperazine 27:

This compound was prepared in two steps starting from thiohemiaminal 48(25.0 mg, 44.6 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine(+)-12-deoxybionectin A (10). The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 5→30% ethyl acetate indichloromethane) to afford epitrithiodiketopiperazine 27 (11.3 mg,41.8%) as a white solid. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. Based on ¹H NMRanalysis at 20° C. in CDCl₃, the product exists as a 3:7 mixture ofminor:major conformers. Without the intention to be limited by theory,Applicant notes that in some embodiments, upon concentration or inconcentrated solution, the epitrithiodiketopiperazine 27 tends todegrade, thus rendering its isolation and characterization particularlyarduous; one of the by-products has been identified as the correspondingepidithiodiketopiperazine 26. ¹H NMR (600 MHz, CDCl₃, 20° C.): Majorconformer: δ 7.89 (br-s, 1H, N_(1′)H), 7.81 (d, J=8.1, 1H, C₈H), 7.53(d, J=7.5, 2H, SO₂Ph-o-H), 7.41 (app-ddd, J=2.3, 6.5, 8.1, 1H, C₇H),7.37 (t, J=7.7, 1H, SO₂Ph-p-H), 7.33 (d, J=8.1, 1H, C_(8′)H), 7.19 (dd,J=6.9, 7.9, 1H, C_(7′)H), 7.16-7.12 (m, 2H, C₅H+C₆H), 7.09 (dd, J=7.8,8.0, 2H, SO₂Ph-m-H), 6.96 (dd, J=7.4, 7.7, 1H, C_(6′)H), 6.89 (d, J=8.0,1H, C_(5′)H), 6.56 (s, 1H, C₂H), 6.25 (d, J=2.5, 1H, C_(2′)H), 4.91 (s,1H, C₁₅H), 3.83 (d, J=15.2, 1H, C₁₂H_(a)), 3.21 (s, 3H, C₁₇H₃), 2.84 (d,J=15.2, 1H, C₁₂H_(b)). Minor conformer: δ 7.77 (br-s, 1H, N₁H), 7.68 (d,J=8.0, 1H, C₈H), 7.38-7.34 (m, 2H, C_(5′)H+C_(8′)H), 7.34-7.32 (m, 1H,C₇H), 7.27-7.22 (m, 2H, C₅H+SO₂Ph-p-H), 7.22-7.19 (m, 2H, SO₂Ph-o-H),7.19-7.16 (m, 1H, C₆H), 7.15-7.12 (m, 1H, C_(6′)H), 6.98-6.94 (m, 1H,C_(7′)H), 6.95 (s, 1H, C₂H), 6.92-6.86 (m, 2H, SO₂Ph-m-H), 5.88 (d,J=2.6, 1H, C_(2′)H), 5.26 (s, 1H, C₁₅H), 3.62 (d, J=15.1, 1H, C₁₂H_(a)),3.03 (s, 3H, C₁₇H₃), 2.85 (d, J=15.1, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz,CDCl₃, 20° C.): Major conformer: δ 168.2 (C₁₃), 162.6 (C₁₆), 142.0 (C₉),137.7 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 135.3 (C₄), 133.1 (SO₂Ph-p-C),130.2 (C₇), 128.6 (SO₂Ph-m-C), 127.5 (SO₂Ph-o-C), 125.8 (C₆), 124.9(C₅), 124.2 (C_(4′)), 124.0 (C_(2′)), 123.0 (C_(7′)), 120.5 (C_(6′)),118.9 (C_(5′)), 118.8 (C₈), 113.8 (C_(3′)), 112.0 (C_(8′)), 84.4 (C₂),79.5 (C₁₁), 67.2 (C₁₅), 53.7 (C₃), 48.8 (C₁₂), 32.8 (C₁₇). Minorconformer: δ 169.9 (C₁₃), 161.5 (C₁₆), 141.2 (C₉), 138.1 (SO₂Ph-ipso-C),137.1 (C_(9′)), 136.6 (C₄), 132.9 (SO₂Ph-p-C), 129.7 (C₇), 128.2(SO₂Ph-m-C), 127.4 (SO₂Ph-o-C), 126.5 (C₆), 124.7 (C₅), 124.2 (C_(2′)),123.9 (C_(4′)), 123.2 (C_(7′)), 120.8 (C_(6′)), 119.4 (C₈), 118.7(C_(5′)), 114.2 (C_(3′)), 112.0 (C_(8′)), 85.4 (C₂), 75.0 (C₁₁), 71.4(C₁₅), 54.1 (C₃), 46.3 (C₁₂), 33.2 (C₁₇). FTIR (thin film) cm⁻¹: 3394(br-m), 3017 (w), 2922 (w), 2852 (w), 1699 (s), 1460 (m), 1364 (m), 1236(w), 1169 (m), 1082 (m), 1049 (w), 750 (m). HRMS (ESI) (m/z): calc'd forC₂₈H₂₃N₄O₄S₄ [M+H]⁺: 607.0597, found 607.0611; calc'd for C₂₈H₂₂N₄NaO₄S₄[M+Na]⁺: 629.0416, found 629.0435. TLC (10% ethyl acetate indichloromethane), Rf: 0.46 (UV, CAM).

C3-(Indol-3′-yl) Epitetrathiodiketopiperazine 28:

The compound was prepared in two steps starting from thiohemiaminal 48(49.3 mg, 88.0 μmol) using the methodology developed to access thecorresponding C3-(indol-3′-yl) epidithiodiketopiperazine(+)-12-deoxybionectin A (10). The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 5→30% acetate indichloromethane) to afford epitetrathiodiketopiperazine 28 (25.0 mg,44.4%) as a white solid. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. Without theintention to be limited by theory, Applicant notes that in someembodiments, the isolation and purification ofepitetrathiodiketopiperazine 28 were complicated by its instability insolution. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.92 (br-s, 1H, N_(1′)H),7.69 (d, J=7.8, 2H, SO₂Ph-o-H), 7.58 (d, J=8.1, 1H, C₈H), 7.40 (t,J=7.4, 1H, SO₂Ph-p-H), 7.34 (d, J=8.2, 1H, C_(8′)H), 7.31 (app-t, J=7.8,1H, C₇H), 7.22-7.16 (m, 4H, C₅H+C_(7′)H+SO₂Ph-m-H), 7.11 (app-t, J=7.4,1H, C₆H), 7.04 (d, J=7.8, 1H, C_(5′)H), 7.01 (dd, J=7.1, 7.7, 1H,C_(6′)H), 6.95 (s, 1H, C₂H), 6.45 (d, J=2.2, 1H, C_(2′)H), 5.23 (s, 1H,C₁₅H), 3.47 (d, J=14.8, 1H, C₁₂H_(a)), 3.06 (s, 3H, C₁₇H₃), 3.03 (d,J=14.8, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 168.2 (C₁₃),162.8 (C₁₆), 141.8 (C₉), 138.5 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 136.4(C₄), 133.2 (SO₂Ph-p-C), 129.7 (C₇), 128.8 (SO₂Ph-m-C), 127.7(SO₂Ph-o-C), 125.7 (C₆), 124.6 (C₅), 124.3 (C_(4′)), 123.0 (C_(2′)),123.0 (C_(7′)), 120.7 (C_(6′)), 118.8 (C_(5′)), 117.3 (C₈), 115.8(C_(3′)), 112.0 (C_(8′)), 85.2 (C₂), 76.0 (C₁₁), 68.3 (C₁₅), 53.6 (C₃),49.1 (C₁₂), 32.5 (C₁₇). FTIR (thin film) cm⁻¹: 3395 (br-w), 3061 (w),2924 (w), 2853 (w), 1690 (s), 1458 (w), 1382 (m), 1240 (w), 1170 (m),1023 (w), 734 (m), 591 (m). HRMS (ESI) (m/z): calc'd for C₂₈H₂₂N₄NaO₄S₅[M+Na]⁺: 661.0137, found 661.0120. TLC (10% ethyl acetate indichloromethane), Rf: 0.30 (UV, I₂, CAM).

C3-(N-Boc-indol-3′-yl) bis(benzylthioether) 43:

Trifluoroacetic acid (4 mL) was slowly added via syringe to a stirredsolution of diol (−)-56 (70.0 mg, 128.6 μmol, 1 equiv) and benzylmercaptan (BnSH, 600 μL, 5.12 mmol, 39.7 equiv) in anhydrous nitroethane(5 mL) at 23° C. After 3 h, the reaction mixture was diluted with ethylacetate (100 mL) and slowly poured into saturated aqueous sodiumhydrogenocarbonate solution (40 mL) at 23° C. The organic layer wassequentially washed with water (3×40 mL) and saturated aqueous sodiumchloride solution (25 mL). The combined aqueous layers were extractedwith ethyl acetate (2×30 mL). The combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 10→40% ethyl acetate inhexanes) to afford the bis(benzylthioether) S22 (77.8 mg, 79.9%) as apale yellow oil. {A minor diastereomer was also isolated from thisreaction (13.0 mg, 13.3%)}.

S22:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.85 (d, J=8.0, 1H, C_(8′)H), 7.75(d, J=7.4, 1H, C₈H), 7.41 (t, J=7.5, 1H, SO₂Ph-p-H), 7.38-7.27 (m, 7H),7.27-7.23 (m, 1H), 7.22-7.15 (m, 4H), 7.18 (app-t, J=7.5, 2H,SO₂Ph-m-H), 7.14-7.10 (m, 2H, C₆H+C_(6′)H), 7.10-7.05 (m, 2H), 6.80-6.76(m, 2H), 6.71 (s, 1H, C₂H), 6.41 (d, J=2.5, 1H, C_(2′)H), 4.48 (s, 1H,C₁₅H), 4.06 (d, J=12.9, 1H, C₁₉H_(a)), 3.81 (d, J=13.6, 1H, C₂₄H_(a)),3.79 (d, J=12.8, 1H, C₁₉H_(b)), 3.76 (d, J=13.7, 1H, C₂₄H_(b)), 3.39 (d,J=14.4, 1H, C₁₂H_(a)), 2.83 (d, J=14.4, 1H, C₁₂H_(b)), 2.53 (s, 3H,C₁₇H₃). MS (ESI) (m/z): [M+H]⁺: 757.56, [M+Na]⁺: 779.60, [M+K]⁺: 795.55.TLC (50% ethyl acetate in hexanes), Rf: 0.40 (UV, CAM).

4-Dimethylaminopyridine (DMAP, 8.0 mg, 65.5 μmol, 0.83 equiv) was addedas a solid to a solution of bis(benzylthioether) S22 (60.0 mg, 79.3μmol, 1 equiv) and di-tert-butyl dicarbonate (Boc₂O, 60.0 mg, 275 μmol,3.47 equiv) in anhydrous acetonitrile (4 mL) at 23° C. After 2 h,another portion of DMAP (2.5 mg, 20.5 μmol, 0.26 equiv) was added. After1 h, the reaction mixture was diluted with ethyl acetate (60 mL). Theresulting mixture was sequentially washed with aqueous 5% citric acidsolution (30 mL), water (2×20 mL), and saturated aqueous sodium chloridesolution (20 mL). The organic layer was dried over anhydrous sodiumsulfate, was filtered, and was concentrated under reduced pressure. Theresulting residue was purified by flash column chromatography (eluent:gradient, 10→50% ethyl acetate in hexanes) to afford the N-Boc-indoleadduct 43 (47.0 mg, 69.2%) as a colorless oil. Structural assignmentswere made with additional information from gCOSY, HSQC, gHMBC, and NOESYdata. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.03 (br-s, 1H, C_(8′)H), 7.79(d, J=8.2, 1H, C₈H), 7.47 (d, J=7.4, 2H, C₂₁H), 7.45-7.39 (m, 3H,C₇H+SO₂Ph-o-H), 7.37 (dd, J=7.5, 7.6, 2H, C₂₂H), 7.32-7.28 (m, 2H,C_(7′)H+C₂₁H), 7.24 (dd, J=7.4, 7.5, 1H, C₂₈H), 7.22-7.17 (m, 3H,C₂₆H+SO₂Ph-p-H), 7.17 (app-t, J=7.5, 1H, C₆H), 7.11 (app-t, J=7.6, 1H,C_(6′)H), 7.01 (d, J=7.5, 1H, C₅H), 7.00-6.92 (m, 5H,C_(5′)H+C₂₇H+SO₂Ph-m-H), 6.68 (s, 1H, C₂H), 6.51 (br-s, 1H, C_(2′)H),4.47 (s, 1H, C₁₅H), 3.96 (d, J=14.0, 1H, C₁₉H_(a)), 3.85 (d, J=14.0, 1H,C19H_(b)), 3.70 (d, J=12.1, 1H, C₂₄H_(a)), 3.51 (d, J=12.1, 1H,C₂₄H_(b)), 3.17 (d, J=14.7, 1H, C₁₂H_(a)), 2.86 (d, J=14.7, 1H,C₁₂H_(b)), 2.57 (s, 3H, C₁₇H₃), 1.66 (s, 9H, OC(CH₃)₃). ¹³C NMR (150MHz, CDCl₃, 20° C.): δ 165.2 (C₁₃), 163.4 (C₁₆), 140.9 (C_(carbamate)),142.1 (C₉), 138.3 (C_(9′)), 137.3 (SO₂Ph-ipso-C), 136.0 (C₄), 136.0(C₂₅), 135.7 (C₂₀), 132.7 (SO₂Ph-p-C), 129.9 (C₂₁), 129.7 (C₂₆), 129.7(SO₂Ph-m-C), 129.5 (C₇), 128.9 (C₂₂), 128.5 (SO₂Ph-o-C), 128.4 (C₂₇),127.8 (C₂₃), 127.4 (C_(4′)), 127.2 (C₂₈), 126.0 (C₆), 125.1 (C_(7′)),124.7 (C_(2′)), 124.1 (C₅), 123.3 (C_(6′)), 120.0 (C_(3′)), 119.2(C_(5′)), 119.1 (C₈), 115.9 (C_(8′)), 84.4 (OC(CH₃)₃), 83.6 (C₂), 70.6(C₁₁), 63.4 (C₁₅), 53.2 (C₃), 45.5 (C₁₂), 37.5 (C₁₉), 37.0 (C₂₄), 31.5(C₁₇), 28.4 (OC(CH₃)₃). FTIR (thin film) cm⁻¹: 3214 (br-w), 3062 (w),3027 (w), 2979 (w), 2930 (w), 2856 (w), 1734 (s), 1696 (s), 1668 (s),1476 (m), 1454 (s), 1373 (s), 1270 (s), 1235 (s), 1171 (s), 1158 (s),1097 (m), 1026 (m), 754 (s), 703 (m). HRMS (ESI) (m/z): calc'd forC₄₇H₄₄N₄NaO₆S₃ [M+Na]⁺: 879.2315, found 879.2303. TLC (30% ethyl acetatein hexanes), Rf: 0.33 (UV, CAM).

C3-(N-Boc-Indol-3′-yl) Epidithiodiketopiperazine 24:

A solution of DMAP in anhydrous dichloromethane (0.17 M, 25 μL, 2.5 mol%) was added via syringe to a solution of epidithiodiketopiperazine 26(98.3 mg, 171 μmol, 1 equiv) and di-tert-butyl dicarbonate (77.6 mg, 355μmol, 2.08 equiv) in anhydrous dichloromethane (20 mL) at 23° C. After 2h, another portion of DMAP solution (25 μL, 2.5 mol %) was added. After5 h, the reaction mixture was diluted with ethyl acetate (100 mL). Theresulting mixture was sequentially washed with aqueous 5% citric acidsolution (50 mL), water (2×50 mL), and saturated aqueous sodium chloridesolution (30 mL). The organic layer was dried over anhydrous sodiumsulfate, was filtered, and was concentrated under reduced pressure. Theresulting residue was purified by flash column chromatography (eluent:gradient, 30→60% ethyl acetate in hexanes) to afford theN-Boc-epidithiodiketopiperazine 24 (93.3 mg, 80.9%) as a pale yellowoil. Structural assignments were made with additional information fromgCOSY, HSQC, and gHMBC data. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.05(br-s, 1H, C_(8′)H), 7.85 (d, J=8.1, 1H, C₈H), 7.48 (app-dt, J=4.5, 8.1,1H, C₇H), 7.38 (app-dt, J=1.3, 7.7, 1H, SO₂Ph-p-H), 7.55 (d, J=7.1, 1H,C₅H), 7.34-7.30 (m, 2H, C₅H+C₆H), 7.28 (dd, J=7.1, 7.3, 1H, C_(7′)H),7.17 (app-t, J=7.4, 1H, C_(6′)H), 7.13 (d, J=7.6, 2H, SO₂Ph-o-H), 6.82(dd, J=7.6, 8.1, 2H, SO₂Ph-m-H), 6.55 (s, 1H, C₂H), 6.18 (br-s, 1H,C_(2′)H), 5.29 (s, 1H, C₁₅H), 3.88 (d, J=15.6, 1H, C₁₂H_(a)), 3.17 (s,3H, C₁₇H₃), 2.67 (d, J=15.6, 1H, C₁₂H_(b)), 1.66 (s, 9H, OC(CH₃)₃). ¹³CNMR (150 MHz, CDCl₃, 20° C.): δ 165.1 (C₁₃), 160.3 (C₁₆), 149.2(C_(carbamate)), 141.0 (C₉), 137.5 (SO₂Ph-ipso-C), 137.5 (C_(9′)), 135.9(C₄), 132.8 (SO₂Ph-p-C), 130.3 (C₇), 128.1 (SO₂Ph-m-C), 127.1(SO₂Ph-o-C), 126.7 (C₆), 125.6 (C_(4′)), 125.4 (C₅), 124.5 (C_(2′)),123.6 (C₇), 123.6 (C_(6′)), 120.1 (C_(5′)), 119.0 (C₈), 118.5 (C_(3′)),116.0 (C_(8′)), 84.6 (OC(CH₃)₃), 84.1 (C₂), 74.4 (C₁₁), 68.5 (C₁₅), 55.2(C₃), 42.2 (C₁₂), 32.3 (C₁₇), 28.4 (OC(CH₃)₃). FTIR (thin film) cm⁻¹:2978 (w), 2929 (w), 1733 (s), 1677 (m), 1454 (m), 1371 (s), 1256 (m),1157 (s), 1092 (m), 751 (s). HRMS (ESI) (m/z): calc'd for C₃₃H₃₀N₄NaO₆S₃[M+Na]⁺: 697.1220, found 697.1231. TLC (50% ethyl acetate in hexanes),Rf: 0.39 (UV, I₂, CAM).

C3-(N-Boc-Indol-3′-yl) bis(S-MOM)ether 40:

Sodium borohydride (50.0 mg, 1.32 mmol, 6.06 equiv) was added as a solidto a solution of epidithiodiketopiperazine 24 (147 mg, 218 μmol, 1equiv) in anhydrous tetrahydrofuran (15 mL) and anhydrous methanol (60.0μL) at 23° C. After 2 h, chloromethyl methyl ether (MOMCl, 500 μL, 1.42mmol, 30.4 equiv) was added to the reaction mixture. After 1 h,triethylamine (200 μL, 1.42 mmol, 6.53 equiv) was added to the reactionmixture. After 4 h, the white reaction mixture was diluted with ethylacetate (100 mL) and washed with saturated aqueous ammonium chloridesolution (30 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL). The combined organic layers were washed sequentially withwater (2×30 mL) and saturated aqueous sodium chloride solution (20 mL),were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 30→70% ethylacetate in hexanes) to afford the bis(S-MOM) derivative S23 (123 mg,73.4%) as a colorless oil.

S23:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.03 (br-s, 1H, C_(8′)H), 7.84(app-dd, J=0.5, 8.2, 1H, C₈H), 7.53 (br-d, J=7.4, 2H, SO₂Ph-o-H), 7.38(app-ddd, J=1.5, 7.4, 8.2, 1H, C₇H), 7.30 (app-t, J=7.6, 1H, C_(7′)H),7.27 (app-dt, J=0.9, 8.3, 1H, C₆H), 7.19 (app-dd, J=0.8, 7.5, 1H, C₅H),7.15 (app-dt, J=0.9, 7.4, 1H, C_(6′)H), 7.05 (dd, J=7.7, 7.8, 2H,SO₂Ph-m-H), 7.00 (dd, 0.1=7.4, 7.6, 2H, SO₂Ph-p-H), 6.73 (d, J=7.8, 1H,C_(5′)H), 6.72 (s, 1H, C₂H), 6.65 (s, 1H, C_(2′)H), 5.21 (d, J=11.8, 1H,C₂₁H_(a)), 5.08 (app-d, J=12.7, 1H, C₁₉H_(a)), 4.95 (s, 1H, C₁₅H), 4.46(d, J=11.8, 1H, C₂₁H_(b)), 4.30 (d, J=12.7, 1H, C₁₉H_(b)), 3.46 (s, 3H,C₂₂H₃), 3.39 (d, J=14.7, 1H, C₁₂H_(a)), 3.23 (d, J=14.7, 1H, C₁₂H_(b)),3.08 (s, 3H, C₁₇H₃), 2.92 (s, 3H, C₂₀H₃), 1.66 (s, 9H, OC(CH₃)₃). TLC(50% ethyl acetate in hexanes), Rf: 0.49 (UV, CAM).

Trifluoroacetic acid (2 mL) was added to a solution of the N-Boc-indoleS23 (6.1 mg, 7.8 μmol, 1 equiv) in anhydrous dichloromethane (5 mL) at0° C. After 30 min, the ice-water bath was removed, and the solution wasallowed to warm to 23° C. After 3 h, the reaction mixture was dilutedwith ethyl acetate (50 mL) and slowly poured into saturated aqueoussodium hydrogenocarbonate solution (25 mL). The organic layer wassequentially washed with water (3×15 mL) and saturated aqueous sodiumchloride solution (15 mL). The combined aqueous layers were extractedwith ethyl acetate (2×20 mL). The combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 10→40% ethyl acetate inhexanes) to afford the bis(S-MOM)ether 40 (4.3 mg. 81%) as a pale yellowoil. Structural assignments were made with additional information fromgCOSY, HSQC, gHMBC, and NOESY data. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ7.87 (br-s, 1H, C_(1′)H), 7.79 (d, J=8.2, 1H, C₈H), 7.72 (d, J=7.7, 2H,SO₂Ph-o-H), 7.41 (app-dd, J=7.4, 7.5, 1H, SO₂Ph-p-H), 7.32 (app-dt,J=0.9, 7.8, 1H, C₇H), 7.29 (d, J=8.1, 1H, C_(8′)H), 7.19 (dd, J=7.7,8.0, 2H, SO₂Ph-m-H), 7.14 (app-t, J=7.5, 1H, C_(7′)H), 7.14 (d, J=7.4,1H, C₅H), 7.08 (dd, J=7.4, 7.7, 1H, C₆H), 6.83 (dd, J=7.4, 7.8, 1H,C_(6′)H), 6.70 (d, J=8.0, 1H, C_(5′)H), 6.68 (s, 1H, C₂H), 6.51 (d,J=2.5, 1H, C_(2′)H), 5.17 (d, J=11.8, 1H, C₂₁H_(a)), 5.11 (d, J=12.7,1H, C₁₉H_(a)), 4.91 (s, 1H, C₁₅H), 4.44 (d, J=11.8, 1H, C₂₁H_(b)), 4.35(d, J=12.7, 1H, C₁₉H_(b)), 3.51 (d, J=14.7, 1H, C₁₂H_(a)), 3.45 (s, 3H,C₂₂H₃), 3.29 (d, J=14.7, 1H, C₁₂H_(b)), 3.07 (s, 3H, C₁₇H₃), 2.93 (s,3H, C₂₀H₃). ¹³C NMR (150 MHz. CDCl₃, 20° C.): δ 165.7 (C₁₃), 163.2(C₁₆), 141.6 (C₉), 138.4 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 136.4 (C₄),133.0 (SO₂Ph-p-C), 129.0 (C₇), 128.8 (SO₂Ph-m-C), 127.5 (SO₂Ph-o-C),125.2 (C₆), 125.0 (C₅), 124.5 (C_(4′)), 122.9 (C_(2′)), 122.7 (C_(4′)),120.4 (C_(6′)), 119.1 (C_(5′)), 117.0 (C₈), 116.5 (C_(3′)), 111.7(C_(8′)), 84.6 (C₂), 76.5 (C₂₁), 75.5 (C₁₉), 70.5 (C₁₁), 64.9 (C₁₅),56.8 (C₂₀), 56.6 (C₂₂), 53.7 (C₃), 49.2 (C₁₂), 32.3 (C₁₇). FTIR (thinfilm) cm⁻¹: 3390 (w), 3004 (w), 2927 (w), 2823 (w), 1693 (s), 1666 (s),1461 (m), 1392 (s), 1364 (s), 1312 (m), 1265 (w), 1235 (w), 1181 (s),1084 (s), 751 (s). HRMS (ESI) (m/z): calc'd for C₃₂H₃₂N₄NaO₆S₃ [M+Na]⁺:687.1376, found: 687.1378. TLC (50% ethyl acetate in hexanes), Rf: 0.38(UV, CAM).

C3-(Indol-3′-yl) Bis(S-MOM)Ether 41:

A 20×150 mm Pyrex tube was sequentially charged with bis(S-MOM)ether S23(92.2 mg, 121 μmol, 1 equiv), 1-ascorbic acid (310 mg, 1.76 mmol, 14.6equiv), sodium 1-ascorbate (380 mg, 1.92 mmol, 15.9 equiv), and1,4-dimethoxynaphthalene (1.25 g, 6.64 mmol, 55.1 equiv), and themixture was placed under an argon atmosphere. A solution of water inacetonitrile (20% v/v, 24 mL) that was purged with argon for 15 min at23° C. was transferred to the flask via cannula. The system wasvigorously stirred under an argon atmosphere and irradiated with aRayonet photoreactor equipped with 16 lamps emitting at 350 nm at 25° C.After 2.5 h, the lamps were turned off, and the reaction mixture wasdiluted with ethyl acetate (100 mL) and diethyl ether (50 mL). Theresulting solution was sequentially washed with saturated aqueous sodiumhydrogenocarbonate solution (50 mL), water (2×40 mL), and saturatedaqueous sodium chloride solution (40 mL). The aqueous layer wasextracted with ethyl acetate (2×50 mL). The combined organic layers weredried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 20→60% ethylacetate in hexanes) to afford aniline S24 (61.7 mg, 81.9%) as a paleyellow oil.

S24:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.11 (br-s, 1H, N_(1′)H), 7.46 (br-s,1H, C_(8′)H), 7.37 (d, J=7.9, 1H, C_(5′)H), 7.26 (app-t, J=7.6, 1H,C_(7′)H), 7.10 (d, J=7.2, 1H, C₅H), 7.13-7.08 (m, 3H, C₆H+C₇H+C₈H),6.74-6.67 (m, 2H, C_(2′)H+C_(6′)H), 6.04 (s, 1H, C₂H), 5.19 (d, J=11.7,1H, C₂₁H_(a)), 5.16 (d, J=12.6, 1H, C₁₉H_(a)), 4.90 (s, 1H, C₁₅H), 4.52(d, J=11.7, 1H, C₂₁H_(b)), 4.31 (d, J=12.6, 1H, C₁₉H_(b)), 3.55 (d,J=14.1, 1H, C₁₂H_(a)), 3.45 (d, J=14.1, 1H, C₁₂H_(b)), 3.47 (s, 3H,C₂₂H), 3.06 (s, 3H, C₁₇H₃), 2.91 (s, 3H, C₂₀H₃), 1.65 (s, 9H, OC(CH₃)).HRMS (ESI) (m/z): calc'd for C₃₁H₃₆N₄NaO₆S₂ [M+Na]⁺: 647.1968, found:647.1976. TLC (50% ethyl acetate in hexanes), Rf: 0.74 (UV, CAM).

Trifluoroacetic acid (2 mL) was added to a solution of the N-Boc-indoleS24 (6.0 mg, 9.6 μmol, 1 equiv) in anhydrous dichloromethane (5 mL) at0° C. After 30 min, the ice-water bath was removed, and the solution wasallowed to warm to 23° C. After 3 h, the reaction mixture was dilutedwith ethyl acetate (50 mL) and slowly poured into saturated aqueoussodium hydrogenocarbonate solution (25 mL) at 23° C. The organic layerwas sequentially washed with water (3×15 mL) and saturated aqueoussodium chloride solution (15 mL). The combined aqueous layers wereextracted with ethyl acetate (2×20 mL). The combined organic layers weredried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 20→60% ethylacetate in hexanes) to afford the bis(S-MOM)ether 41 (4.6 mg, 91%) as acolorless oil. Structural assignments were made with additionalinformation from gCOSY, HSQC, gHMBC, and NOESY data. ¹H NMR (600 MHz,CDCl₃, 20° C.): δ 7.98 (br-s, 1H, N_(1′)H), 7.38 (d, J=8.0, 1H,C_(5′)H), 7.32 (d, J=8.2, 1H, C_(8′)H), 7.16 (app-t, J=7.2, 1H,C_(7′)H), 7.14 (d, J=6.9, 1H, C₅H), 7.10 (app-dt, J=1.0, 7.8, 1H, C₇H),7.02 (app-t, J=7.2, 1H, C_(6′)H), 7.01 (d, J=2.7, 1H, C_(2′)H), 6.73(dd, J=7.3, 7.5, 1H, C₆H), 6.71 (d, J=7.8, 1H, C₈H), 6.05 (s, 1H, C₂H),5.22 (d, J=11.7, 1H, C₂₁H_(a)), 5.13 (d, J=12.6, 1H, C₁₉H_(a)), 4.93 (s,1H, C₁₅H), 4.53 (d, J=11.7, 1H, C₂₁H_(b)), 4.34 (d, J=12.6, 1H,C₁₉H_(b)), 3.48 (s, 2H, C₁₂H), 3.48 (s, 3H, C₂₂H₃), 3.07 (s, 3H, C₁₇H₃),2.95 (s, 3H, C₂₀H₃). The resonance for N₁H was not observed. ¹³C NMR(150 MHz, CDCl₃, 20° C.): δ 166.1 (C₁₃), 165.4 (C₁₆), 148.5 (C₉), 137.4(C_(9′)), 132.8 (C₄), 128.6 (C₇), 125.3 (C_(4′)), 124.9 (C₅), 122.6(C_(7′)), 121.6 (C_(2′)), 120.2 (C_(6′)), 119.9 (C_(5′)), 119.6 (C₆),119.4 (C_(3′)), 111.6 (C_(8′)), 109.3 (C₈), 82.6 (C₂), 77.0 (C₂₁), 75.7(C₁₉), 69.3 (C₁₁), 65.1 (C₁₅), 57.0 (C₂₀), 56.5 (C₂₂), 54.3 (C₃), 48.0(C₁₂), 32.0 (C₁₇). FTIR (thin film) cm⁻¹: 3394 (br-w), 3013 (w), 2928(w), 2823 (w), 1693 (s), 1669 (s), 1461 (m), 1393 (m), 1363 (m), 1265(w), 1180 (s), 752 (s). HRMS (ESI) (m/z): calc'd for C₂₆H₂₈N₄NaO₄S₂[M+Na]⁺: 547.1444, found: 547.1434. TLC (50% ethyl acetate in hexanes),Rf: 0.43 (UV, CAM).

C3-(N-Boc-Indol-3′-yl) bis(S-MEM)ether 42 and C3-(N-Boc-indol-3′-yl)S15-MEM ether 47:

Sodium borohydride (9.8 mg, 250 μmol, 3.6 equiv) was added as a solid toa solution of epidithiodiketopiperazine 24 (47.0 mg, 69.6 μmol, 1 equiv)in anhydrous tetrahydrofuran (8 mL) and anhydrous methanol (50 μL) at23° C. After 80 min, 2-methoxyethoxymethyl chloride (MEMCl, 300 μL, 2.63mmol, 37.7 equiv) followed by triethylamine (400 μL, 2.85 mmol, 40.9equiv) were added to the reaction mixture. After 12 h, the yellowreaction mixture was partitioned between aqueous 5% citric acid solution(30 mL) and ethyl acetate (80 mL). The isolated organic layer was washedsequentially with water (2×30 mL) and saturated aqueous sodium chloridesolution (20 mL), was dried over anhydrous sodium sulfate, was filtered,and was concentrated under reduced pressure. The residue was purified byflash column chromatography on silica gel (eluent: gradient, 10→25%ethyl acetate in dichloromethane) to afford the bis(S-MEM)ether adduct42 (57.6 mg, 80.2%) and the S15-MEM-adduct 47 (10.0 mg, 18.8%) ascolorless oils. Structural assignments were made with additionalinformation from gCOSY, HSQC, gHMBC, and NOESY data.

C3-(N-Boc-indol-3′-yl) bis(S-MEM)ether 42:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.02 (br-s, 1H, C_(8′)H), 7.83 (d,J=8.2, 1H, C₈H), 7.56 (br-d, J=6.2, 2H, SO₂Ph-o-H), 7.36 (dd, J=7.7,8.0, 1H, C₇H), 7.32 (t, J=7.3, 1H, SO₂Ph-p-H), 7.24 (d, J=7.8, 1H,C_(5′)H), 7.16 (d, J=7.4, 1H, C₅H), 7.10 (app-t, J=7.6, 1H, C₆H), 7.08(app-t, J=7.5, 2H, SO₂Ph-m-H), 6.96 (app-t, J=7.5, 1H, C_(6′)H), 6.76(s, 1H, C_(2′)H), 6.67-6.61 (m, 1H, C_(7′)H), 6.62 (s, 1H, C₂H), 5.21(d, J=12.0, 1H, C₂₃H_(a)), 5.13 (d, J=12.8, 1H, C₁₉H_(a)), 5.00 (s, 1H,C₁₅H), 4.63 (d, J=12.0, 1H, C₂₃H_(b)), 4.47 (d, J=12.8, 1H, C₁₉H_(b)),4.00-3.94 (m, 1H, C₂₄H_(a)), 3.67-3.64 (m, 2H, C₂₄H_(b)+C₂₅H_(a)),3.61-3.56 (m, 1H, C₂₅H_(b)), 3.41 (d, J=14.9, 1H, C₁₂H_(a)), 3.39 (s,3H, C26H₃), 3.38-3.33 (m, 2H, C₂₁H), 3.31 (s, 3H, C₂₂H₃), 3.28-3.23 (m,1H, C₂₀H_(a)), 3.23 (d, J=14.9, 1H, C₁₂H_(b)), 3.20-3.14 (m, 1H,C₁₂H_(b)), 3.09 (s, 3H, C₁₇H₃), 1.69 (s, 9H, (OC(CH₃)₃). ¹³C NMR (150MHz, CDCl₃, 20° C.): δ 165.3 (C₁₃), 163.1 (C₁₆), 149.3 (C_(carbamate)),141.7 (C₉), 137.9 (SO₂Ph-ipso-C), 136.3 (C_(9′)), 135.7 (C₄), 133.0(SO₂Ph-p-C), 129.3 (C₇), 128.6 (SO₂Ph-nm-C), 127.4 (SO₂Ph-o-C), 127.2(C_(4′)), 125.5 (C₆), 125.0 (C₅), 124.9 (C_(5′)), 124.5 (C_(7′)), 123.2(C_(6′)), 120.3 (C_(3′)), 119.1 (C_(2′)), 117.9 (C₈), 115.7 (C_(8′)),84.4 (OC(CH₃)₃), 83.8 (C₂), 75.2 (C₂₃), 74.0 (C₁₉), 71.6 (C₂₅), 71.6(C₂₁), 70.3 (C₁₁), 68.2 (C₂₀), 68.1 (C₂₄), 64.9 (C₁₅), 59.3 (C₂₂), 59.2(C₂₆), 53.3 (C₃), 48.6 (C₁₂), 32.2 (C₁₇), 28.4 (OC(CH₃)₃). FTIR (thinfilm) cm⁻¹: 2920 (m), 2851 (m), 1734 (s), 1699 (s), 1668 (s), 1454 (s),1373 (s), 1310 (m), 1272 (m), 1158 (s), 1088 (s), 1025 (m), 752 (s).HRMS (ESI) (m/z): calc'd for C₄₁H₄₈N₄NaO₁₀S₃ [M+Na]⁺: 875.2425, found:875.2411. TLC (20% ethyl acetate in dichloromethane), Rf: 0.44 (UV, I₂,CAM).

C3-(N-Boc-Indol-3′-yl) S15-MEM ether 47:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.04 (br-s, 1H, C_(8′)H), 7.85 (d,J=8.1, 1H, C₈H), 7.45 (app-dt, J=1.0, 8.0, 1H, C₇H), 7.38 (br-d, J=6.2,2H, SO₂Ph-o-H), 7.31 (app-t, J=7.8, 1H, C_(7′)H), 7.29-7.21 (m, 3H,C₅H+C₆H+SO₂Ph-p-H), 7.12 (dd, J=7.4, 7.6, 1H, C_(6′)H), 6.95 (app-t,J=7.7, 2H, SO₂Ph-m-H), 6.91 (d, J=7.8, 1H, C_(5′)H), 6.67 (s, 1H, C₂H),6.53 (s, 1H, C_(2′)H), 5.25 (d, J=11.9, 1H, C₂₃H_(a)), 5.09 (s, 1H,C₁₅H), 4.71 (d, J=11.9, 1H, C₂₃H_(b)), 4.00-3.96 (m, 1H, C₂₄H_(a)),3.70-3.62 (m, 2H, C₂₄H_(b)+C₂₅H_(a)), 3.62-3.58 (m, 1H, C₂₅H_(b)), 3.43(d, J=14.6, 1H, C₁₂H_(a)), 3.40 (s, 3H, C₂₆H₃), 3.13 (s, 3H, C₁₇H₃),2.87 (d, J=14.6, 1H, C₁₂H_(b)), 1.66 (s, 9H, (OC(CH₃)₃). ¹³C NMR (150MHz, CDCl₃, 20° C.): δ 167.5 (C₁₃), 162.2 (C₁₆), 149.2 (C_(carbamate)),142.2 (C₉), 137.8 (SO₂Ph-ipso-C), 135.6 (C_(9′)), 135.6 (C₄), 132.8(SO₂Ph-p-C), 129.9 (C₇), 128.3 (SO₂Ph-m-C), 127.3 (SO₂Ph-o-C), 126.9(C_(4′)), 126.3 (C₆), 125.2 (C_(7′)), 124.9 (C₅), 124.8 (C_(2′)), 123.4(C_(6′)), 119.6 (C_(3′)), 119.3 (C₈), 119.0 (C_(5′)), 115.9 (C_(8′)),84.5 (OC(CH₃)₃), 83.8 (C₂), 74.0 (C_(2′)), 71.6 (C₂₅), 68.4 (C₂₄), 68.1(C₁₁), 64.3 (C₁₅), 59.3 (C₂₆), 53.4 (C₃), 51.2 (C₁₂), 32.7 (C₁₇), 28.4(OC(CH₃)₃). FTIR (thin film) cm⁻¹: 2978 (w), 2922 (w), 1734 (m), 1697(m), 1454 (m), 1372 (s), 1272 (w), 1235 (w), 1157 (m), 1091 (m), 752(s). HRMS (ESI) (m/z): calc'd for C₃₇H₄₀N₄NaO₈S₃ [M+Na]⁺: 787.1900,found: 787.1897. TLC (20% ethyl acetate in dichloromethane), Rf: 0.24(UV, I₂, CAM).

C3-(Indol-3′-yl) dithiepanethione 36:

Sodium borohydride (4.9 mg, 0.13 mmol, 3.4 equiv) was added as a solidto a solution of epidithiodiketopiperazine 26 (22.0 mg, 38.3 μmol, 1equiv) in anhydrous tetrahydrofuran (5 mL) and anhydrous methanol (50μL) at 23° C. After 45 min, the reaction mixture was diluted with ethylacetate (40 mL) and washed with saturated aqueous ammonium chloridesolution (20 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL) and the combined organic layers were washed sequentially withwater (2×20 mL) and saturated aqueous sodium chloride solution (20 mL),were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure to afford the hexacyclic bisthiolS25 that was used in the next step without further purification.1,1′-Thiocarbonyldiimidazole (TCDI, 108 mg, 606 μmol, 15.8 equiv) wasadded as a solid to the solution of bisthiol S25 in anhydrousdichloromethane (6 mL) at 23° C. After 22 h, the volatiles were removedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: gradient, 5→25% ethyl acetate indichloromethane) to afford the dithiepanethione 36 (8.4 mg, 34%) as apale yellow oil. Structural assignments were made with additionalinformation from gCOSY, HSQC, and gHMBC data. Without the intention tobe limited by theory, Applicant notes that in some embodiments, uponconcentration or in concentrated solution, the dithiepanethione 36 tendsto degrade, thus rendering its isolation and characterizationparticularly arduous. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.86 (br-s, 1H,N_(1′)H), 7.75 (d, J=8.2, 1H, C₈H), 7.49 (app-dd, J=1.0, 8.3, 2H,SO₂Ph-o-H), 7.43-7.36 (m, 2H, C₇H+SO₂Ph-p-H), 7.35 (d, J=8.1, 1H,C_(8′)H), 7.23-7.17 (m, 3H, C₅H+C₆H+C_(7′)H), 7.10 (dd, J=7.6, 8.1, 2H,SO₂Ph-m-H), 6.98 (app-dt, J=0.5, 7.5, 1H, C_(6′)H), 6.89 (d, J=8.0, 1H,C_(5′)H), 6.64 (s, 1H, C₂H), 6.36 (d, J=2.5, 1H, C_(2′)H), 5.05 (s, 1H,C₁₅H), 3.96 (d, J=15.6, 1H, C₁₂H_(a)), 3.18 (s, 3H, C₁₇H₃), 2.80 (d,J=15.6, 1H, C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 214.3 (C₁₉),164.3 (C₁₃), 159.4 (C₁₆), 140.9 (C₉), 137.5 (SO₂Ph-ipso-C), 137.3(C_(9′)), 135.1 (C₄), 133.2 (SO₂Ph-p-C), 130.2 (C₇), 128.7 (SO₂Ph-m-C),127.4 (SO₂Ph-o-C), 126.2 (C₆), 124.6 (C₅), 124.2 (C_(4′)), 124.1(C_(2′)), 123.2 (C_(7′)), 120.7 (C_(6′)), 118.8 (C_(5′)), 118.5 (C₈),113.8 (C_(3′)), 112.0 (C_(8′)), 85.3 (C₂), 75.3 (C₁₁), 69.5 (C₁₅), 54.6(C₃), 45.8 (C₁₂), 32.7 (C₁₇). FTIR (thin film) cm⁻¹: 3393 (br-w), 2921(m), 2851 (w), 1703 (s), 1459 (m), 1361 (m), 1168 (m), 1089 (w), 1016(w), 907 (w), 733 (m). HRMS (ESI) (m/z): calc'd for C₂₉H₂₃N₄O₄S₄ [M+H]⁺:619.0597, found 619.0609; calc'd for C₂₉H₂₂N₄NaO₄S₄ [M+Na]⁺: 641.0416,found 641.0424. TLC (20% ethyl acetate in dichloromethane), Rf: 0.68(UV, I₂, CAM).

C3-(Indol-3′-yl) dithiocarbonate 37:

Sodium borohydride (4.9 mg, 0.13 mmol, 3.3 equiv) was added as a solidto a solution of epidithiodiketopiperazine 26 (22.6 mg, 39.3 μmol, 1equiv) in anhydrous tetrahydrofuran (5 mL) and anhydrous methanol (50μL) at 23° C. After 45 min, the reaction mixture was diluted with ethylacetate (40 mL) and washed with saturated aqueous ammonium chloridesolution (20 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL) and the combined organic layers were washed sequentially withwater (2×20 mL) and saturated aqueous sodium chloride solution (20 mL),were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure to afford the hexacyclic bisthiolS25 that was used in the next step without further purification.1,1′-Carbonyldiimidazole (CDI, 80.0 mg, 493 μmol, 12.0 equiv) was addedas a solid to the solution of bisthiol S25 in anhydrous dichloromethane(10 mL) at 23° C. After 24 h, the volatiles were removed under reducedpressure. The residue was purified by flash column chromatography onsilica gel (eluent: gradient, 5→20% ethyl acetate in dichloromethane) toafford the dithiocarbonate 37 along with epidithiodiketopiperazine 26.Both compounds were separated by preparative HPLC [Waters X-Bridgepreparative HPLC column, C18, 5 μm, 19×250 mm; 20.0 mL/min; gradient,20→90% acetonitrile in water, 20 min; t_(R)(37)=15.35 min,t_(R)(26)=14.50 min] to afford 37 (2.0 mg, 8%) as a pale yellow oil.Epidithiodiketopiperazine 26 was also recovered (2.9 mg, 12%).Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.84 (br-s, 1H,N_(1′)H), 7.78 (d, J=8.2, 1H, C₈H), 7.45 (d, J=8.4, 2H, SO₂Ph-o-H), 7.41(app-ddd, J=1.6, 7.3, 7.7, 1H, C₇H), 7.37 (app-dt, J=1.0, 6.4, 1H,SO₂Ph-p-H), 7.35 (d, J=8.2, 1H, C_(8′)H), 7.21 (app-ddd, 0.1=0.8, 7.2,7.6, 1H, C_(7′)H), 7.19 (app-dt, J=0.9, 7.3, 1H, C₆H), 7.16 (app-dd,J=1.1, 7.6, 1H, C₅H), 7.06 (dd, J=7.6, 8.3, 2H, SO₂Ph-m-H), 6.99(app-dt, J=0.7, 7.5, 1H, C_(6′)H), 6.90 (d, J=7.9, 1H, C_(5′)H), 6.64(s, 1H, C₂H), 6.26 (d, J=2.6, 1H, C_(2′)H), 5.17 (s, 1H, C₁₅H), 3.92 (d,J=15.5, 1H, C₁₂H_(a)), 3.20 (s, 3H, C₁₇H₃), 2.78 (d, J=15.5, 1H,C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 185.4 (C₁₉), 165.0 (C₁₃),160.0 (C₁₆), 141.0 (C₉), 137.3 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 135.2(C₄), 133.2 (SO₂Ph-p-C), 130.2 (C₇), 128.7 (SO₂Ph-m-C), 127.5(SO₂Ph-o-C), 126.2 (C₆), 124.6 (C₅), 124.1 (C_(4′)), 124.1 (C_(2′)),123.2 (C_(7′)), 120.7 (C_(6′)), 118.7 (C_(5′)), 118.7 (C₈), 113.8(C_(3′)), 112.0 (C_(8′)), 85.3 (C₂), 72.6 (C₁₁), 66.6 (C₁₅), 54.5 (C₃),46.5 (C₁₂), 32.6 (C₁₇). FTIR (thin film) cm⁻¹: 3396 (br-m), 2924 (m),2853 (w), 1696 (m), 1460 (m), 1383 (m), 1169 (m), 1091 (w), 1051 (w),735 (m). HRMS (ESI) (m/z): calc'd for C₂₉H₂₂N₄NaO₅S₃ [M+Na]⁺: 625.0645,found 625.0652. TLC (20% ethyl acetate in dichloromethane), Rf: 0.57(UV, I₂, CAM).

C3-(Indol-3′-yl) dithioacetal 38:

Sodium borohydride (15.0 mg, 0.400 mmol, 9.88 equiv) was added as asolid to a solution of epidithiodiketopiperazine 26 (23.1 mg. 40.2 μmol,1 equiv) in anhydrous THF (5 mL) and diiodomethane (0.2 mL) at 0° C.under an argon atmosphere ((a) Cook, K. M.; Hilton, S. T.; Mecinović,J.; Motherwell, W. B.; Figg, W. D.; Schofield, C. J. J. Biol. Chem.2009, 284, 26831. (b) Poisel, H.; Schmidt, U. Chem. Ber. 1971, 104,1714). After 5 min, anhydrous methanol (50 μL) was added. After 50 min,the reaction mixture was partitioned between aqueous hydrochloric acidsolution (1 N, 25 mL) and ethyl acetate (80 mL). The aqueous layer wasextracted with ethyl acetate (2×20 mL), and the combined organic layerswere washed sequentially with water (2×30 mL) and saturated aqueoussodium chloride solution (30 mL), were dried over anhydrous sodiumsulfate, were filtered, and were concentrated under reduced pressure.The residue was purified by flash column chromatography on silica gel(eluent: gradient, 5→20% ethyl acetate in dichloromethane) to afforddithioacetal 38 (10.8 mg, 45.6%) as a colorless oil. Structuralassignments were made with additional information from gCOSY, HSQC, andgHMBC data. Based on ¹H NMR analysis at 20° C. in CDCl₃, the productexists as a 1:4 mixture of minor:major conformers. ¹H NMR (600 MHz,CDCl₃, 20° C.): Major conformer: δ 7.89 (br-s, 1H, N_(1′)H), 7.87 (d,J=8.2, 1H, C₈H), 7.65 (d, J=7.7, 2H, SO₂Ph-o-H), 7.43 (app-dd, J=7.4,7.5, 1H, SO₂Ph-p-H), 7.37 (app-ddd, J=1.3, 7.4, 8.2, 1H, C₇H), 7.31 (d,J=8.2, 1H, C_(8′)H), 7.15 (dd, J=7.7, 7.9, 2H, SO₂Ph-m-H), 7.14 (app-t,J=7.3, 1H, C_(7′)H), 7.10 (app-t, J=7.5, 1H, C₆H), 7.02 (app-dd,0.1=0.4, 7.4, 1H, C₅H), 6.81 (app-dd, J=7.4, 7.7, 1H, C_(6′)H), 6.57 (s,1H, C₂H), 6.51 (d, J=8.0, 1H, C_(5′)H), 6.43 (d, J=2.4, 1H, C_(2′)H),4.86 (s, 1H, C₁₅H), 4.55 (d, J=14.8, 1H, C₁₉H_(a)), 3.86 (d, J=14.9, 1H,C₁₂H_(a)), 3.71 (d, J=14.8, 1H, C₁₉H_(b)), 3.08 (s, 3H, C₁₇H₃), 2.70 (d,J=14.9, 1H, C₁₂H_(b)). Minor conformer: δ 7.78 (d, J=8.3, 1H, C₈H), 7.78(br-s, 1H, N_(1′)H), 7.44-7.40 (m, 1H, C₇H), 7.35 (d, J=7.7, 2H,SO₂Ph-o-H), 7.32-7.29 (m, 1H, C_(7′)H), 7.28 (d, J=8.3, 1H, C₅H),7.27-7.21 (m, C_(5′)H SO₂Ph-p-H), 7.20 (d, J=8.0, 1H, C_(5′)H),7.12-7.07 (m, 2H, C₆H+C_(6′)H), 6.93 (dd, J=7.7, 8.0, 2H, SO₂Ph-m-H),6.57 (s, 1H, C₂H), 5.97 (d, J=2.5, 1H, C_(2′)H), 5.26 (s, 1H, C₁₅H),4.01 (d, J=15.6, 1H, C₁₉H_(a)), 3.56 (d, J=14.9, 1H, C₁₉H_(b)), 3.16 (s,3H, C₁₇H₃), 3.11 (d, J=15.8, 1H, C₁₂H_(a)), 2.72 (d, J=15.8, 1H,C₁₂H_(b)). ¹³C NMR (150 MHz, CDCl₃, 20° C.): Major conformer: δ 167.5(C₁₃), 161.7 (C₁₆), 140.5 (C₉), 137.3 (C_(9′)), 136.6 (SO₂Ph-ipso-C),136.1 (C₄), 133.4 (SO₂Ph-p-C), 129.5 (C₇), 128.8 (SO₂Ph-m-C), 128.0(SO₂Ph-o-C), 125.7 (C₆), 124.6 (C₅), 124.4 (C_(4′)), 124.1 (C_(2′)),122.9 (C_(7′)), 120.4 (C_(6′)), 119.0 (C_(5′)), 117.8 (C₈), 113.9(C_(3′)), 111.8 (C_(8′)), 85.4 (C₂), 70.6 (C₁₁), 65.2 (C₁₅), 54.4 (C₃),48.1 (C₁₂), 32.7 (C₁₇), 31.7 (C₁₉). Minor conformer: δ 165.3 (C₁₃),160.5 (C₁₆), 140.8 (C₉), 137.6 (C_(9′)), 137.2 (SO₂Ph-ipso-C), 136.6(C₄), 133.0 (SO₂Ph-p-C), 129.9 (C₇), 128.4 (SO₂Ph-m-C), 127.3(SO₂Ph-o-C), 126.1 (C₆), 124.8 (C₅), 124.4 (C_(4′)), 124.2 (C_(2′)),123.2 (C_(7′)), 120.8 (C_(6′)), 119.1 (C_(5′)), 118.8 (C₈), 114.2(C_(3′)), 111.9 (C_(8′)), 84.8 (C₂), 74.5 (C₁₁), 68.5 (C₁₅), 55.7 (C₃),42.6 (C₁₂), 32.7 (C₁₇), 32.3 (C₁₉). FTIR (thin film) cm⁻¹: 3392 (br-m),3059 (w), 2977 (w), 1690 (s), 1451 (w), 1361 (m), 1266 (w), 1170 (m),1090 (w), 1022 (m), 736 (m). HRMS (ESI) (m/z): calc'd for C₂₉H₂₄N₄NaO₄S₃[M+Na]⁺: 611.0852, found 611.0850. TLC (10% ethyl acetate indichloromethane), Rf: 0.40 (UV, I₂, CAM).

C3-(Indol-3′-yl) epimonothiodiketopiperazine 25

(Cherblanc, F.; Lo, Y.-P.; De Gussem, E.; Alcazar-Fuoli, L.; Bignell,E.; He, Y.; Chapman-Rothe, N.; Bultinck, P.; Herrebout, W. A.; Brown,R.; Rzepa, H. S.; Fuchter, M. J. Chem.—Eur. J. 2011, 17, 11868):Triethylphosphite (10.0 μL, 58.4 μmol, 21.4 equiv) was added to thesolution of epidithiodiketopiperazine 26 (8.6 mg, 15 μmol, 1 equiv) inanhydrous tetrahydrofuran (4 mL) at 23° C. After 6 h, the reactionmixture was diluted sequentially with saturated aqueous ammoniumchloride solution (20 mL) and ethyl acetate (60 mL). The organic layerwas washed with water (2×15 mL) and saturated aqueous sodium chloridesolution (15 mL), was dried over anhydrous sodium sulfate, was filtered,and was concentrated under reduced pressure. The residue was purified byflash column chromatography on silica gel (eluent: gradient, 2→8% ethylacetate in dichloromethane) to afford the epimonothiodiketopiperazine 25(5.1 mg, 63%) as a white solid. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. Limitedsolubility of epimonothiodiketopiperazine 25 was observed in CH₂Cl₂,CHCl₃, EtOAc, MeOH, DMSO. ¹H NMR (600 MHz, acetone-d₆, 20° C.): δ 9.95(br-s, 1H, N_(1′)H), 7.64 (d, J=8.1, 1H, C₈H), 7.59 (d, J=7.8, 1H,C_(5′)H), 7.52 (d, J=7.3, 1H, C₅H), 7.48 (d, J=8.0, 1H, C_(8′)H), 7.44(app-dt, J=1.1, 7.8, 1H, C₇H), 7.32 (app-tt, J=0.9, 7.4, 1H, SO₂Ph-p-H),7.28 (app-dt, J=0.9, 7.6, 1H, C₆H), 7.26 (app-dt, J=0.9, 7.6, 1H,C_(7′)H), 7.20 (app-dt, J=0.8, 7.5, 1H, C_(6′)H), 6.98 (app-dd, J=1.0,8.3, 2H, SO₂Ph-o-H), 6.90 (app-t, J=7.5, 2H, SO₂Ph-m-H), 6.19 (s, 1H,C₂H), 5.67 (d, J=2.6, 1H, C_(2′)H), 5.17 (s, 1H, C₁₅H), 3.70 (d, J=15.4,1H, C₁₂H_(a)), 3.11 (s, 3H, C₁₇H₃), 2.84 (d, J=15.4, 1H, C₁₂H_(b)). ¹³CNMR (150 MHz, acetone-d₆, 20° C.): δ 173.9 (C₁₃), 171.6 (C₁₆), 141.4(C₉), 138.7 (SO₂Ph-ipso-C), 138.7 (C_(9′)), 137.9 (C₄), 133.9(SO₂Ph-p-C), 130.2 (C₇), 129.0 (SO₂Ph-m-C), 127.2 (SO₂Ph-o-C), 126.4(C₆), 125.7 (C₅), 125.3 (C_(2′)), 124.9 (C_(4′)), 123.0 (C_(7′)), 120.6(C_(6′)), 119.0 (C_(5′)), 118.6 (C₈), 115.3 (C_(3′)), 113.1 (C_(8′)),83.3 (C₂), 81.9 (C₁₁), 73.0 (C₁₅), 59.4 (C₃), 35.3 (C₁₂), 31.4 (C₁₇).FTIR (thin film) cm⁻¹: 3357 (br-w), 3059 (w), 2919 (w), 2851 (w), 1740(s), 1713 (s), 1457 (m), 1358 (m), 1261 (w), 1169 (m), 1086 (w), 971(w), 737 (s), 685 (m). HRMS (ESI) (m/z): calc'd for C₂₈H₂₂N₄NaO₄S₂[M+Na]⁺: 565.0975, found 565.0971. TLC (10% ethyl acetate indichloromethane), Rf: 0.76 (UV, I₂, CAM).

C3-(Indol-3′-yl) Bisthioacetate 44:

Sodium borohydride (4.9 mg, 0.13 mmol, 3.3 equiv) was added as a solidto a solution of epidithiodiketopiperazine 26 (22.6 mg, 39.3 μmol, 1equiv) in anhydrous tetrahydrofuran (5 mL) and anhydrous methanol (50μL) at 23° C. After 45 min, the reaction mixture was diluted with ethylacetate (40 mL) and washed with saturated aqueous ammonium chloridesolution (20 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL), and the combined organic layers were washed sequentially withwater (2×20 mL) and saturated aqueous sodium chloride solution (20 mL),were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure to afford the hexacyclic bisthiolS25 that was used in the next step without further purification. Acetylchloride (200 μL, 2.80 mmol, 71.3 equiv) was added to the solution ofbisthiol S25 in anhydrous dichloromethane (6 mL) and anhydrous pyridine(300 μL, 3.72 mmol, 94.7 equiv) at 23° C. After 4 h, the reactionmixture was diluted with ethyl acetate (60 mL) and washed with aqueous5% citric acid solution (2×20 mL). The aqueous layer was extracted withethyl acetate (2×20 mL), and the combined organic layers were washedsequentially with water (2×20 mL), saturated aqueous sodiumhydrogenocarbonate solution (20 mL), water (20 mL), and saturatedaqueous sodium chloride solution (20 mL), were dried over anhydroussodium sulfate, were filtered, and were concentrated under reducedpressure. The residue was purified by flash column chromatography onsilica gel (eluent: gradient, 5→20% ethyl acetate in dichloromethane) toafford bisthioacetate 44 (17.0 mg, 62.7%) as a pale yellow oil.Structural assignments were made with additional information from gCOSY,HSQC, and gHMBC data. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.01 (br-s, 1H,N_(1′)H), 7.78 (d, J=8.1, 1H, C₈H), 7.75 (d, J=8.2, 2H, SO₂Ph-o-H), 7.42(app-dt, J=1.0, 7.5, 1H, SO₂Ph-p-H), 7.37-7.33 (m, 1H, C₇H), 7.29 (d,J=8.2, 1H, C_(8′)H), 7.21 (dd, J=7.6, 8.3, 2H, SO₂Ph-m-H), 7.12 (dd,J=7.4, 8.0, 1H, C_(7′)H), 7.08-7.03 (m, 2H, C₅H+C₆H), 6.81 (dd, J=7.2,7.9, 1H, C_(6′)H), 6.72 (s, 1H, C₂H), 6.58 (d, J=8.0, 1H, C_(5′)H), 6.55(d, J=2.5, 1H, C_(2′)H), 6.09 (s, 1H, C₁₅H), 3.44 (d, J=14.7, 1H,C₁₂H_(a)), 3.26 (d, J=14.7, 1H, C₁₂H_(b)), 2.98 (s, 3H, C₁₇H₃), 2.48 (s,3H, C₂₂H₃), 2.06 (s, 3H, C₂₀H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ194.0 (C₂₁), 193.9 (C₁₉), 165.1 (C₁₃), 161.9 (C₁₆), 142.0 (C₉), 137.7(SO₂Ph-ipso-C), 137.3 (C_(9′)), 135.2 (C₄), 133.3 (SO₂Ph-p-C), 129.6(C₇), 129.0 (SO₂Ph-m-C), 127.5 (SO₂Ph-o-C), 125.1 (C₆), 124.9 (C₅),124.4 (C_(4′)), 122.9 (C_(2′)), 122.8 (C_(7′)), 120.4 (C_(6′)), 118.7(C_(5′)), 116.6 (C₈), 115.7 (C_(3′)), 111.9 (C_(8′)), 84.8 (C₂), 73.3(C₁₁), 63.5 (C₁₅), 53.6 (C₃), 49.3 (C₁₂), 32.3 (C₁₇), 30.6 (C₂₀), 30.5(C₂₂). FTIR (thin film) cm⁻¹: 3395 (br-m), 3063 (w), 2923 (m), 2852 (w),1699 (br-s), 1459 (m), 1368 (m), 1311 (w), 1172 (m), 1121 (m), 1093 (m),1025 (w), 954 (w), 734 (m). HRMS (ESI) (m/z): calc'd for C₃₂H₂₈N₄NaO₆S₃[M+Na]⁺: 683.1063, found 683.1047. TLC (20% ethyl acetate indichloromethane), Rf: 0.52 (UV, I₂, CAM).

C3-(Indol-3′-yl)N-(thiomethyl) bis(methyldisulfane) 45

((a) Gilow, H. M.; Brown, C. S.; Copeland, J. N.; Kelly, K. E. J.Heterocyclic Chem. 1991, 28, 1025. (b) Kim, J. K.; Caserio, M. C. J.Org. Chem. 1979, 44, 1897. (c) Kharasch, N.; Parker, A. J. J. Org. Chem.1959, 24, 1029): Sodium borohydride (3.7 mg, 0.10 mmol, 5.2 equiv) wasadded as a solid to a solution of epidithiodiketopiperazine 26 (10.8 mg,18.8 μmol, 1 equiv) in anhydrous tetrahydrofuran (5 mL) and anhydrousmethanol (50 μL) at 23° C. After 45 min, the reaction mixture wasdiluted with ethyl acetate (40 mL) and washed with saturated aqueousammonium chloride solution (20 mL). The aqueous layer was extracted withethyl acetate (2×20 mL), and the combined organic layers were washedsequentially with water (2×20 mL) and saturated aqueous sodium chloridesolution (20 mL), were dried over anhydrous sodium sulfate, werefiltered, and were concentrated under reduced pressure to afford thehexacyclic bisthiol S25 that was used in the next step without furtherpurification. A solution of methanesulfinyl chloride (Douglass, I. B.;Norton, R. V.; Farah, B. S. Org. Synth. 1960, 40, 62) in dichloromethane(1.6 M, 250 μL, 402 μmol, 21.4 equiv) was added to the solution ofbisthiol S25 in anhydrous dichloromethane (5 mL) and anhydrous pyridine(100 μL, 1.24 mmol, 66.0 equiv) at 0° C. After 10 min, the ice-waterbath was removed, and the yellow solution was allowed to warm to 23° C.After 2 h, the reaction mixture was diluted sequentially with saturatedaqueous ammonium chloride solution (20 mL) and ethyl acetate (60 mL).The organic layer was sequentially washed with saturated aqueousammonium chloride solution (20 mL), water (2×15 mL), and saturatedaqueous sodium chloride solution (15 mL). The aqueous layer wasextracted with ethyl acetate (2×20 mL). The combined organic layers weredried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 5→30% ethylacetate in hexanes) to afford the N-thiomethyl bis(methyldisulfane) 45(6.5 mg, 49%) as a colorless oil. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. ¹H NMR (600MHz, CDCl₃, 20° C.): δ 7.74 (d, J=8.1, 1H, C₈H), 7.57 (d, J=8.2, 1H,C_(8′)H), 7.42-7.37 (m, 1H, C₇H), 7.39 (d, J=8.2, 2H, SO₂Ph-o-H), 7.35(app-dt, J=1.0, 7.4, 1H, SO₂Ph-p-H), 7.31 (app-dt, J=0.8, 7.7, 1H,C_(7′)H), 7.27 (app-dt, J=1.0, 7.6, 1H, C₅H), 7.21 (app-dt, J=0.8, 7.5,1H, C₆H), 7.09 (app-dt, J=0.7, 7.5, 1H, C_(6′)H), 7.01 (d, J=7.9, 1H,C_(5′)H), 6.97 (dd, J=7.6, 8.2, 2H, SO₂Ph-m-H), 6.71 (s, 1H, C₂H), 6.08(s, 1H, C_(2′)H), 5.02 (s, 1H, C₁₅H), 3.29 (d, J=15.0, 1H, C₁₂H_(a)),3.25 (d, J=15.0, 1H, C₁₂H_(b)), 3.17 (s, 3H, C₁₇H₃), 2.67 (s, 3H,C₂₀H₃), 2.50 (s, 3H, C₂₁H₃), 2.29 (s, 3H, C₁₉H₃). ¹³C NMR (150 MHz,CDCl₃, 20° C.): δ 165.3 (C₁₃), 162.5 (C₁₆), 142.0 (C₉), 141.2 (C_(9′)),137.8 (SO₂Ph-ipso-C), 136.0 (C₄), 133.0 (C_(2′)), 132.9 (SO₂Ph-p-C),129.7 (C₇), 128.4 (SO₂Ph-m-C), 127.3 (SO₂Ph-o-C), 125.9 (C₆), 125.8(C_(4′)), 124.6 (C₅), 123.7 (C_(7′)), 121.6 (C_(6′)), 119.1 (C_(5′)),118.8 (C₈), 117.6 (C_(3′)), 111.7 (C_(8′)), 84.8 (C₂), 79.2 (C₁₅), 73.9(C₁₁), 53.5 (C₃), 46.0 (C₁₂), 32.7 (C₁₇), 24.4 (C₂₀), 24.0 (C₂₁), 23.3(C₁₉). FTIR (thin film) cm⁻¹: 2925 (w), 1699 (s), 1458 (m), 1359 (m),1231 (w), 1168 (m), 1091 (w), 749 (m). HRMS (ESI) (m/z): calc'd forC₃₁H₃₀N₄NaO₄S₆ [M+Na]⁺: 737.0484, found 737.0469. TLC (50% ethyl acetatein hexanes), Rf: 0.60 (UV, I₂, CAM).

C3-(Indol-3′-yl) bis(methyldisulfane) 46:

Sodium borohydride (4.8 mg, 0.13 mmol, 3.7 equiv) was added as a solidto a solution of epidithiodiketopiperazine 26 (19.5 mg, 33.9 μmol, 1equiv) in anhydrous tetrahydrofuran (5 mL) and anhydrous methanol (50μL) at 23° C. After 45 min, the reaction mixture was diluted with ethylacetate (40 mL) and washed with saturated aqueous ammonium chloridesolution (20 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL), and the combined organic layers were washed sequentially withwater (2×20 mL) and saturated aqueous sodium chloride solution (20 mL),were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure to afford the hexacyclic bisthiolS25 that was used in the next step without further purification.Dimethyldisulfide (200 μL, 2.23 mmol, 65.7 equiv) (Dubs, P.; Stuessi, R.Helv. Chim. Acta 1976, 59, 1307) was added to the solution of bisthiolS25 in anhydrous tetrahydrofuran (6 mL) at 23° C. After 19 h, thevolatiles were removed under reduced pressure. The residue was purifiedby flash column chromatography on silica gel (eluent: gradient, 5→10%ethyl acetate in dichloromethane) to afford the bis(methyldisulfane) 46(9.3 mg, 41%) as a colorless oil. Structural assignments were made withadditional information from gCOSY, HSQC, and gHMBC data. ¹H NMR (600MHz, CDCl₃, 20° C.): δ 7.85 (br-s, 1H, N_(1′)H), 7.70 (d, J=8.1, 1H,C₈H), 7.52 (d, J=7.4, 2H, SO₂Ph-o-H), 7.36 (app-dt, J=1.3, 7.8, 1H,C₇H), 7.33 (t, J=7.5, 1H, SO₂Ph-p-H), 7.32 (d, J=8.2, 1H, C_(8′)H), 7.24(app-dd, J=0.8, 7.5, 1H, C₅H), 7.19 (app-ddd, J=2.4, 5.8, 8.2, 1H,C_(7′)H), 7.16 (app-dt, J=0.8, 7.5, 1H, C₆H), 7.07 (app-dt, J=0.5, 7.9,2H, SO₂Ph-m-H), 7.01-6.96 (m, 2H, C_(5′)H+C_(6′)H), 6.76 (s, 1H, C₂H),6.28 (d, J=2.6, 1H, C_(2′)H), 5.00 (s, 1H, C₁₅H), 3.38 (d, J=15.0, 1H,C₁₂H_(a)), 3.26 (d, J=15.0, 1H, C₁₂H_(b)), 3.17 (s, 3H, C₁₇H₃), 2.64 (s,3H, C₂₀H₃), 2.29 (s, 3H, C₁₉H₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ165.4 (C₁₃), 162.6 (C₁₆), 141.8 (C₉), 138.2 (SO₂Ph-ipso-C), 137.3(C_(9′)), 136.4 (C₄), 133.0 (SO₂Ph-p-C), 129.5 (C₇), 128.6 (SO₂Ph-m-C),127.4 (SO₂Ph-o-C), 125.7 (C₆), 124.6 (C₅), 124.2 (C_(4′)), 123.3(C_(2′)), 122.9 (C_(7′)), 120.5 (C_(6′)), 118.9 (C_(5′)), 118.3 (C₈),115.7 (C_(3′)), 111.9 (C_(8′)), 85.2 (C₂), 79.2 (C₁₅), 74.0 (C₁₁), 53.6(C₃), 46.4 (C₁₂), 32.7 (C₁₇), 24.4 (C₂₀), 23.4 (C₁₉). FTIR (thin film)cm⁻¹: 3392 (br-m), 3060 (w), 2921 (w), 1685 (s), 1459 (m), 1391 (m),1266 (w), 1169 (m), 1092 (w), 1022 (w), 736 (m). HRMS (ESI) (m/z):calc'd for C₃₀H₂N₄NaO₄S₅ [M+Na]⁺: 691.0606, found 691.0613. TLC (20%ethyl acetate in dichloromethane), Rf: 0.67 (UV, I₂, CAM).

C3-(Indol-3′-yl)-pyrrolidinoindoline 74:

This compound was prepared in two steps starting from endo-tetracyclicbromide (Kim, J.; Ashenhurst, J. A.; Movassaghi, M. Science 2009, 324,238) (+)-73 (512.5 mg, 10.5 mmol, 1 equiv) using the methodologydeveloped to access the correspondingC3-(5-bromo-N-TIPS-indol-3′-yl)-pyrrolidinoindoline (+)-S12 with DTBMP(339 mg, 1.65 mmol, 1.58 equiv), 5-bromo-1-triisopropylsilyl-1H-indoleS11 (1.92 g, 5.45 mmol, 5.20 equiv)(5-Bromo-1-triisopropylsilyl-1H-indole S11 was prepared in quantitativeyield by silylation of commercially available 5-bromoindole usingtriisopropylsilyl chloride and sodium hydride in tetrahydrofuran. Forpreparation and characterization, see: Brown, D. A.; Mishra, M.; Zhang,S.; Biswas, S.; Parrington, I.; Antonio, T.; Reith, M. E. A.; Dutta, A.K. Bioorg. Med. Chem. 2009, 17, 3923), and silver(I) tetrafluoroborate(600 mg, 3.08 mmol, 2.95 equiv) in anhydrous nitroethane (12 mL). After1 h, saturated aqueous sodium chloride solution (20 mL) was introduced,and the resulting biphasic mixture was vigorously stirred for 30 min at0° C. The reaction mixture was diluted with ethyl acetate (50 mL), wasfiltered through a Celite pad, and the solid was washed with ethylacetate (3×15 mL). The combined filtrates were washed with 5% aqueouscitric acid solution (2×25 mL), water (3×25 mL), and saturated aqueoussodium chloride solution (25 mL). The organic layer was dried overanhydrous sodium sulfate, was filtered, and was concentrated underreduced pressure. The resulting residue was purified by flash columnchromatography (eluent: gradient, 1→10% acetone in dichloromethane) toafford the C3-(5-bromo-N-TIPS-indol-3′-yl)-pyrrolidinoindoline S26 (537mg, 67.4%) as a white foam.

The free indole was accessed in a one-pot two-step procedure using themethodology developed to access the correspondingC3-(indol-3′-yl)-pyrrolidinoindoline (+)-59. The reaction mixture wasfiltered through a pad of Celite. The solids were washed with ethylacetate (3×50 mL). The combined filtrates were concentrated underreduced pressure. The resulting pale yellow solid was diluted in ethylacetate (150 mL) and washed sequentially with an aqueous hydrochloricacid solution (1 N, 2×50 mL), water (2×50 mL), and saturated aqueoussodium chloride solution (40 mL). The organic layer was dried overanhydrous sodium sulfate, was filtered, and was concentrated underreduced pressure to afford the C3-(indol-3′-yl)-pyrrolidinoindoline 74(370 mg, 99.7%) as a white solid that was used in the next step withoutfurther purification.

S26:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.03 (d, J=7.8, 2H, SO₂Ph-o-H), 7.78(d, J=8.4, 1H, C₈H), 7.54 (app-dd, 0.1=7.2, 7.8, 1H, SO₂Ph-p-H), 7.40(app-t, J=7.8, 2H, SO₂Ph-m-H), 7.30 (d, J=8.9, 1H, C_(8′)H), 7.28(app-dt, J=1.0, 7.9, 1H, C₇H), 7.15 (app-dd, J=1.7, 8.8, 1H, C_(7′)H),6.97 (dd, J=7.5, 7.6, 1H, C₆H), 6.95 (s, 1H, C_(2′)H), 6.82 (d, J=7.3,1H, C₅H), 6.50 (br-s, 1H, C_(5′)H), 6.30 (s, 1H, C₂H), 4.44 (dd, J=7.8,9.4, 1H, C₁₁H), 3.97 (q, J=7.1, 1H, C₁₅H), 3.03 (dd, J=7.5, 13.8, 1H,C₁₂H_(a)), 2.99 (s, 3H, C₁₈H₃), 2.88 (dd, J=9.8, 13.8, 1H, C₁₂H_(b)),1.66 (d, J=7.1, 3H, C₁₇H), 1.59 (app-sp, J=7.5, 3H, C_(10′)H), 1.07(app-d, J=5.5, 18H, C_(11′)H). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 169.5(C₁₃), 169.2 (C₁₆), 141.3 (C_(9′)), 139.6 (C₉), 137.1 (SO₂Ph-ipso-C),134.2 (SO₂Ph-p-C), 133.9 (C₄), 130.9 (C_(2′)), 130.3 (C_(4′)), 129.5(C₇), 129.2 (SO₂Ph-m-C), 127.9 (SO₂Ph-o-C), 125.3 (C_(7′)), 124.5 (C₆),123.9 (C₅), 121.9 (C_(5′)), 116.0 (C_(8′)), 115.6 (C₈), 115.1 (C_(3′)),113.5 (C_(6′)), 83.0 (C₂), 59.5 (C₁₁), 57.5 (C₁₅), 55.3 (C₃), 37.8(C₁₂), 29.6 (C₁₈), 18.2 (C₁₁), 14.8 (C₁₁), 12.9 (C_(10′)). TLC (20%/oacetone in dichloromethane), Rf: 0.76 (UV, CAM).

74:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.94 (br-s, 1H, N_(1′)H), 7.74 (d,J=8.2, 1 H, C₈H), 7.46 (d, J=8.2, 2H, SO₂Ph-o-H), 7.35 (app-dt, 0.1=0.9,7.5, 1H, SO₂Ph-p-H), 7.34 (d, J=8.3, 1H, C_(8′)H), 7.29 (dd, J=7.5, 8.1,1H, C₇H), 7.19 (app-dt, J=4.1, 8.2, 1H, C_(7′)H), 7.12 (d, J=7.5, 1H,C₅H), 7.09-7.04 (m, 3H, SO₂Ph-m-H+C₆H), 6.95 (app-d, J=4.0, 2H,C_(5′)H+C_(6′)H), 6.40 (s, 1H, C₂H), 6.09 (d, J=2.0, 1H, C_(2′)H), 4.52(app-t, J=7.8, 1H, C₁₁H), 4.07 (q, J=7.0, 1H, C₁₅H), 3.10 (app-d, J=7.8,2H, C₁₂H), 2.90 (s, 3H, C₁₈H₃), 1.61 (d, J=7.1, 3H, C₁₇H₃). MS (ESI)(m/z): [M+H]⁺: 527.25; [M+Na]⁺: 549.21. TLC (20% acetone indichloromethane), Rf: 0.27 (UV, CAM).

C3-(Indol-3′-yl) Dithiepanethiones 64 and 66:

Freshly prepared bis(pyridine)silver(I) permanganate (Firouzabadi, H.;Vessal, B.; Naderi, M. Tetrahedron Lett. 1982, 23, 1847) (800 mg, 2.08mmol, 5.45 equiv) was added as a solid to a solution of indole adduct 74(201 mg, 382 μmol, 1 equiv) in anhydrous pyridine (5 mL) at 23° C. After2 h, a second portion of bis(pyridine)silver(I) permanganate (600 mg,1.56 mmol, 4.08 equiv) was added. After 2 h, the resulting thick brownsuspension was diluted with saturated aqueous sodium sulfite solution(50 mL) and then with ethyl acetate (160 mL). The resulting mixture waswashed sequentially with water (2×50 mL), aqueous 5% copper sulfatesolution (3×50 mL), and saturated aqueous sodium chloride solution (30mL). The combined aqueous layers were extracted with ethyl acetate(2×100 mL). The combined organic layers were dried over anhydrous sodiumsulfate, were filtered, and were concentrated under reduced pressure.The resulting yellow residue was purified by flash column chromatography(eluent: gradient, 2→20% isopropanol in dichloromethane and hexanes(50%)) to afford the corresponding diols (78.0 mg, 36.5%) as a yellowoil (isolated as a mixture of isomers).

To a yellow solution of potassium trithiocarbonate (250 mg, 1.34 mmol,9.63 equiv) (Stueber, D.; Patterson, D.; Mayne, C. L.; Orendt, A. M.;Grant, D. M.; Parry, R. W. Inorg. Chem. 2001, 40, 1902) in anhydrousdichloromethane (6 mL) and trifluoroacetic acid (4 mL) at 23° C. wasadded a solution of the diol (78.0 mg, 139 μmol, 1 equiv) indichloromethane (1 mL). After 2.5 h, the reaction mixture was dilutedwith ethyl acetate (60 mL) and washed with saturated aqueous sodiumbicarbonate (30 mL). The aqueous layer was extracted with ethyl acetate(2×20 mL) and the combined organic layers were dried over anhydroussodium sulfate, were filtered, and were concentrated under reducedpressure. The resulting orange residue was purified by flash columnchromatography on silica gel (eluent: gradient, 2→8% ethyl acetate indichloromethane) to afford an inseparable mixture of isomeric monomericdithiepanethiones 64 and 66 (55.7 mg, 63.3%, 64:66, 5:1) as a paleyellow solid. Isomers 64 and 66 were separated for the purpose of fulland independent characterization by preparative HPLC [Waters X-Bridgepreparative HPLC column, C18, 5 μm, 19×250 mm; 20.0 mL/min; gradient,30→100% acetonitrile in water, 35 min; t_(R)(64)=21.3 min,t_(R)(66)=23.4 min]. Structural assignments were made using additionalinformation from gCOSY, HSQC, and HMBC experiments.

β-epimer 64:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.81 (br-s, 1H, N_(1′)H), 7.74 (d,J=8.2, 1H, C₈H), 7.44-7.39 (m, 1H, C₇H), 7.37 (app-dd, J=0.7, 7.5, 2H,SO₂Ph-o-H), 7.35 (d, J=8.1, 1H, C_(8′)H), 7.34 (t, J=7.5, 1H,SO₂Ph-p-H), 7.26-7.21 (m, 3H, C5H+C₆H+C_(7′)H), 7.08-7.04 (m, 2H,C_(5′)H+C_(6′)H), 7.02 (app-t, J=7.7, 2H, SO₂Ph-m-H), 6.74 (s, 1H, C₂H),6.20 (d, J=2.5, 1H, C_(2′)H), 3.91 (d, J=15.6, 1H, C₁₂H_(a)), 3.13 (s,3H, C₁₈H₃), 2.86 (d, J=15.6, 1H, C₁₂H_(b)), 2.01 (s, 3H, C₁₇H₃). ¹³C NMR(100 MHz, CDCl₃, 20° C.): δ 215.9 (C₁₉), 165.0 (C₁₃), 161.0 (C₁₆), 141.1(C₉), 137.8 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 135.4 (C₄), 133.1(SO₂Ph-p-C), 130.2 (C₇), 128.5 (SO₂Ph-m-C), 127.3 (SO₂Ph-o-C), 126.3(C₆), 124.7 (C₅), 124.1 (C_(4′)), 124.0 (C_(2′)), 123.2 (C_(7′)), 120.8(C_(6′)), 119.0 (C₈), 118.8 (C_(5′)), 114.1 (C_(3′)), 112.0 (C_(8′)),85.6 (C₂), 75.1 (C₁₁), 73.5 (C₁₅), 54.1 (C₃), 46.4 (C₁₂), 28.7 (C₁₈),20.2 (C₁₇). FTIR (thin film) cm⁻¹: 3397 (br-m), 3061 (w), 1688 (s), 1459(w), 1361 (s), 1241 (w), 1170 (s), 1108 (w), 1001 (m), 908 (w), 734 (m).HRMS (ESI) (m/z): calc'd for C₃₀H₂₅N₄O₄S₄ [M+H]⁺: 633.0753, found633.0744. TLC (50% ethyl acetate in hexanes), Rf: 0.33 (UV, CAM).

α-Epimer 66:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.80 (app-dd, J=1.6, 6.8, 1H,C_(5′)H), 7.72 (d, J=8.0, 1H, C₈H), 7.54 (br-s, 1H, N_(1′)H), 7.40-7.34(m, 3H, C₅H+C₇H+C_(8′)H), 7.34-7.29 (m, 2H, C_(6′)H+C_(7′)H), 7.22(app-t, J=7.5, 1H, C₆H), 7.20 (t, 0.1=7.4, 1H, SO₂Ph-p-H), 7.06 (app-dd,J=0.9, 8.3, 2H, SO₂Ph-o-H), 6.81 (dd, J=7.6, 8.1, 2H, SO₂Ph-m-H), 6.79(s, 1H, C₂H), 5.55 (d, J=2.5, 1H, C_(2′)H), 4.04 (d, J=15.6, 1H,C₁₂H_(a)), 3.11 (d, J=15.6, 1H, C₁₂H_(b)), 2.98 (s, 3H, C₁₈H₃), 2.00 (s,3H, C₁₇H₃). ¹³C NMR (100 MHz, CDCl₃, 20° C.): δ 209.5 (C₁₉), 164.5(C₁₃), 161.1 (C₁₆), 139.3 (C₉), 138.3 (SO₂Ph-ipso-C), 137.3 (C_(9′)),135.8 (C₄), 132.7 (SO₂Ph-p-C), 130.0 (C₇), 128.1 (SO₂Ph-m-C), 127.0(SO₂Ph-o-C), 125.9 (C₆), 125.4 (C₅), 124.9 (C_(2′)), 123.7 (C_(4′)),123.5 (C_(7′)), 121.3 (C_(6′)), 119.1 (C_(5′)), 118.2 (C₈), 114.4(C_(3′)), 112.0 (C_(8′)), 85.4 (C₂), 74.9 (C₁₁), 73.6 (C₁₅), 54.9 (C₃),42.0 (C₁₂), 28.8 (C₁₈), 21.3 (C₁₇). FTIR (thin film) cm⁻¹: 3396 (br-m),2924 (w), 1698 (s), 1458 (m), 1364 (m), 1334 (m), 1251 (w), 1169 (m),1091 (m), 1013 (m), 912 (w), 734 (m). HRMS (ESI) (m/z): calc'd forC₃₀H₂₅N₄O₄S₄ [M+H]⁺: 633.0753, found 633.0767. TLC (50% ethyl acetate inhexanes), Rf: 0.33 (UV, CAM).

C3-(Indol-3′-yl) Epidithiodiketopiperazines 60 and 62:

Ethanolamine (4 mL) was added via syringe to a solution of thebisdithiepanethiones 64 and 66 (33.0 mg, 52.1 μmol, 1 equiv, 64:66, 5:1)in acetone (6 mL) at 23° C. After 45 min, the reaction mixture waspartitioned between ethyl acetate (100 mL) and aqueous hydrochloric acidsolution (1 N, 30 mL). The organic layer was collected, and the aqueouslayer was extracted with ethyl acetate (2×15 mL). A solution ofpotassium triiodide in pyridine (2.5% w/v) was added dropwise to thecombined organic layers until a persistent yellow color was observed.The resulting mixture was washed with aqueous hydrochloric acid (1 N, 20mL), and saturated aqueous sodium chloride solution (20 mL). The organiclayer was dried over anhydrous sodium sulfate, was filtered, and wasconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: gradient, 20→50% ethylacetate in hexanes) to afford an inseparable mixture of isomericmonomeric epidithiodiketopiperazines 60 and 62 (14.8 mg, 48.2%, 60:62,5:1) as a pale yellow solid. Isomers 60 and 62 were separated for thepurpose of full and independent characterization by preparative HPLC[Waters X-Bridge preparative HPLC column, C18, 5 μm, 19×250 mm; 20.0mL/min; gradient, 30→100% acetonitrile in water, 35 min; t_(R)(60)=18.0min, t_(R)(62)=19.7 min]. Structural assignments were made usingadditional information from gCOSY, HSQC, and HMBC experiments.

β-Epimer 60:

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.01 (d, J=7.3, 1H, C_(5′)H), 7.73(d, J=8.0, 1H, C₈H), 7.60 (br-s, 1H, N_(1′)H), 7.41 (d, J=6.6, 1H, C₅H),7.39 (d, J=7.8, 1H, C_(8′)H), 7.43-7.38 (m, 1H, C₇H), 7.38-7.31 (m, 2H,C_(6′)H+C_(7′)H), 7.26-7.21 (m, 2H, C₆H+SO₂Ph-p-H), 7.11 (app-dd, J=0.9,8.3, 2H, SO₂Ph-o-H), 6.85 (dd, J=7.6, 8.1, 2H, SO₂Ph-nm-H), 6.84 (s, 1H,C₂H), 5.58 (d, J=2.5, 1H, C_(2′)H), 4.00 (d, J=15.1, 1H, C₁₂H_(a)), 3.19(d, J=15.1, 1H, C₁₂H_(b)), 2.97 (s, 3H, C₁₈H₃), 2.04 (s, 3H, C₁₇H₃). ¹³CNMR (100 MHz, CDCl₃, 20° C.): δ 166.0 (C₁₃), 161.9 (C₁₆), 140.9 (C₉),137.6 (SO₂Ph-ipso-C), 137.2 (C_(9′)), 136.9 (C₄), 133.0 (SO₂Ph-p-C),129.8 (C₇), 128.3 (SO₂Ph-m-C), 127.2 (SO₂Ph-o-C), 126.1 (C₆), 124.5(C₅), 124.3 (C_(4′)), 124.0 (C_(2′)), 123.1 (C_(7′)), 120.7 (C_(6′)),119.3 (C₈), 118.7 (C_(5′)), 114.1 (C_(3′)), 112.1 (C_(8′)), 85.1 (C₂),73.9 (C₁₅), 73.5 (C₁₁), 55.3 (C₃), 43.0 (C₁₂), 27.8 (C₁₈), 18.4 (C₁₇).FTIR (thin film) cm⁻¹: 3396 (br-m), 3061 (w), 2924 (w), 2851 (w), 1704(s), 1447 (w), 1360 (m), 1332 (s), 1244 (w), 1169 (s), 1109 (m), 1090(m), 910 (w), 735 (s). HRMS (ESI) (m/z): calc'd for C₂₉H₂₅N₄O₄S₃ [M+H]⁺:589.1032, found 589.1043. TLC (50% ethyl acetate in hexanes), Rf: 0.27(UV, CAM).

α-Epimer 62:

¹H NMR (600 MHz. CDCl₃, 20° C.): δ 7.99 (d, J=7.4, 1H, C_(5′)H), 7.70(d, J=8.0, 1H, C₈H), 7.57 (br-s, 1H, N_(1′)H), 7.40-7.34 (m, 3H,C₅H+C₇H+C_(8′)H), 7.34-7.28 (m, 2H, C_(6′)H+C_(7′)H), 7.21 (app-dt,J=1.7, 7.6, 2H, C₆H+SO₂Ph-p-H), 7.08 (app-dd, J=0.9, 8.3, 2H,SO₂Ph-o-H), 6.82 (dd, J=7.6, 8.1, 2H, SO₂Ph-m-H), 6.82 (s, 1H, C₂H),5.55 (d, J=2.5, 1H, C_(2′)H), 3.97 (d, J=15.1, 1H, C₁₂H_(a)), 3.16 (d,J=15.1, 1H, C₁₂H_(b)), 2.94 (s, 3H, C₁₈H₃), 2.01 (s, 3H, C₁₇H₃). ¹³C NMR(100 MHz, CDCl₃, 20° C.): δ 165.9 (C₁₃), 162.6 (C₁₆), 139.5 (C₉), 138.3(SO₂Ph-ipso-C), 137.3 (C_(9′)), 135.6 (C₄), 132.6 (SO₂Ph-p-C), 129.8(C₇), 128.1 (SO₂Ph-m-C), 127.1 (SO₂Ph-o-C), 125.9 (C₆), 125.4 (C₅),124.6 (C_(2′)), 123.9 (C_(4′)), 123.4 (C_(7′)), 121.0 (C_(6′)), 119.2(C_(5′)), 118.4 (C₈), 115.2 (C_(3′)), 111.9 (C_(8′)), 85.0 (C₂), 74.4(C₁₁), 73.8 (C₁₅), 55.9 (C₃), 41.2 (C₁₂), 27.6 (C₁₈), 18.7 (C₁₇). FTIR(thin film) cm⁻¹: 3395 (br-m), 2923 (w), 1701 (s), 1460 (w), 1359 (m),1332 (m), 1247 (w), 1168 (m), 1090 (w), 912 (w), 734 (m). HRMS (ESI)(m/z): calc'd for C₂₉H₂₅N₄O₄S₃ [M+H]⁺: 589.1032, found 589.1037. TLC(50% ethyl acetate in hexanes), Rf: 0.27 (UV, CAM).

Dimeric Bisdithiepanethione 18:

Dimeric tetraol 22 (200 mg, 226 μmol, 1 equiv) was added as a solid to ayellow solution of potassium trithiocarbonate (632 mg, 3.39 mmol, 15.0equiv) in anhydrous dichloromethane (5.1 mL) and trifluoroacetic acid(1.7 mL) at 23° C. After 25 min, the reaction mixture was diluted withdichloromethane (60 mL) and washed with saturated aqueous sodiumbicarbonate (125 mL). The aqueous layer was extracted withdichloromethane (2×30 mL) and the combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure to afford a yellow powder. This powder waspurified by flash column chromatography on silica gel (eluent: 5%acetone in dichloromethane) to afford dimeric bisdithiepanethione 18(88.8 mg, 38.0%) as an orange-yellow solid. ¹H NMR (500 MHz, CDCl₃, 20°C.): δ 7.75-7.65 (m, 2H, C₈H), 7.75-7.65 (m, 4H, SO₂Ph-o-H), 7.53(app-t, J=7.4, 2H, SO₂Ph-p-H), 7.41 (app-t, J=8.0, 4H, SO₂Ph-m-H),7.30-7.14 (m, 6H, C₆H, C₇H, C₅H), 6.86 (s, 2H, C₂H), 3.26 (d, J=14.9,2H, C₁₂H_(a)), 3.09 (d, J=14.9, 2H, C₁₂H_(b)), 3.01 (s, 6H, C₁₈H), 1.68(s, 6H, C₁₇H). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 215.1 (C═S), 164.1(C₁₃), 159.7 (C₁₆), 142.7 (C₉), 141.9 (SO₂Ph-ipso-C), 133.1 (SO₂Ph-p-C),131.3 (C₄), 129.2 (SO₂Ph-m-C), 129.2 (C₆), 125.5 (SO₂Ph-o-C), 125.2(C₇), 124.5 (C₈), 116.1 (C₅), 81.6 (C₂), 73.9 (C₁₁), 73.6 (C₁₅), 59.1(C₃), 44.7 (C₁₂), 28.6 (C₁₈), 19.3 (C₁₇). FTIR (thin film) cm⁻¹: 1715(s), 1691 (s), 1479 (m), 1462 (m), 1447 (m), 1359 (s), 1169 (s), 729(m). HRMS (ESI) (m/z): calc'd for C₄₄H₃₆N₆NaO₈S₈ [M+Na]⁺: 1055.0252,found 1055.0255. [α]_(D) ²⁴: +230 (c 0.19, CHCl_(h)). TLC (5% acetone indichloromethane), Rf: 0.27 (UV, CAM).

Dimeric Epidithiodiketopiperazine 14:

Ethanolamine (500 μL) was added via syringe to a solution of dimericbisdithiepanethione 18 (11.2 mg, 10.8 μmol, 1 equiv) in acetone (500 μL)at 23° C. After 15 min, the reaction mixture was diluted withdichloromethane (30 mL) and aqueous hydrochloric acid solution (1 N, 30mL). The organic layer was collected, and the aqueous layer wasextracted with dichloromethane (2×5 mL). A solution of potassiumtriiodide in pyridine (2.5% w/v) was added dropwise to the combinedorganic layers until a persistent yellow color was observed. Theresulting mixture was washed with aqueous hydrochloric acid (1 N, 30 mL)and the layers were separated. The aqueous layer was extracted withdichloromethane (2×5 mL), and the combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: 5% acetone in dichloromethane) toafford dimeric epidithiodiketopiperazine 14 (3.9 mg, 38%) as a whitesolid. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.85 (dd, J=1.4, 7.3, 4H,SO₂Ph-o-H), 7.68 (d, J=7.5, 2H, C₈H), 7.54 (tt, J=1.2, 7.5, 2H,SO₂Ph-p-H), 7.46 (app-t, J=8.0, 4H, SO₂Ph-m-H), 7.20 (app-dt, J=1.3,7.5, 2H, C₆H), 7.16 (app-dt, J=1.2, 7.5, 2H, C₇H), 7.04 (dd, J=1.0, 7.6,2H, C₅H), 6.83 (s, 2H, C₂H), 3.55 (d, J=15.2, 2H, C₁₂H_(a)), 2.97 (s,6H, C₁₈H), 2.95 (d, J=15.2, 2H, C₁₂H_(b)), 1.62 (s, 6H, C₁₇H). ¹³C NMR(125.8 MHz, CDCl₃, 20° C.): δ 164.9 (C₁₃), 160.8 (C₁₆), 142.5 (C₉),142.4 (SO₂Ph-ipso-C), 132.6 (SO₂Ph-p-C), 130.9 (C₄), 130.6 (C₆), 129.0(SO₂Ph-m-C), 125.7 (SO₂Ph-o-C), 125.2 (C₇), 124.7 (C₈), 116.3 (C₅), 81.9(C₂), 73.8 (C₁₅), 73.4 (C₁₁), 60.5 (C₃), 41.9 (C₁₂), 27.8 (C₁₈), 17.9(C₁₇). FTIR (thin film) cm⁻¹: 1716 (s), 1688 (s), 1480 (m), 1462 (m),1447 (w), 1348 (s), 1168 (m). HRMS (ESI) (m/z): calc'd for C₄₂H₃₇N₆O₈S₆[M+H]⁺: 945.0992, found 945.0968. TLC (5% acetone in dichloromethane),Rf: 0.21 (UV, CAM).

C3-Propyl Dithiepanethiones 65 and 67:

A solution of the tetracyclic diol S27 (228 mg, 470 μmol, 1 equiv) indichloromethane (3.5 mL) was added to a yellow solution of potassiumtrithiocarbonate (438 mg, 2.35 mmol, 5.00 equiv) in anhydrousdichloromethane (7 mL) and trifluoroacetic acid (3 mL) at 23° C. Anadditional portion of trifluoroacetic acid (1.5 mL) was added to thereaction mixture via syringe. After 25 min, the reaction mixture wasdiluted with dichloromethane (60 mL) and washed with saturated aqueoussodium bicarbonate (125 mL). The aqueous layer was extracted withdichloromethane (2×30 mL) and the combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure to yield a yellow powder. This powder waspurified by flash column chromatography on silica gel (eluent: 20%acetone in dichloromethane) to afford diastereomeric dithiepanethiones65 (137 mg, 52.0%) and 67 (38.7 mg, 14.7%) as yellow films.

β-Epimer 65:

¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.72 (d, J=7.5, 2H, SO₂Ph-o-H), 7.52(t, J=7.5, 1H, SO₂Ph-p-H), 7.40 (app-t, J=7.9, 2H, SO₂Ph-m-H), 7.35 (d,J=7.1, 1H, C₈H), 7.29 (app-dt, J=1.7, 8.2, 1H, C₇H), 7.19 (app-dt,J=0.9, 7.7, 1H, C₆H), 7.16 (dd, J=1.4, 7.6, 1H, C₅H), 6.29 (s, 1H, C₂H),3.00 (s, 3H, C₁₈H), 2.98 (d, J=15.1, 1H, C₁₂H_(a)), 2.75 (d, J=15.1, 1H,C₁₂H_(b)), 1.79 (s, 3H, C₁₇H), 1.47-1.31 (m, 2H, CH₂CH₂CH₃), 1.47-1.31(m, 1H, CH₂CH_(a)H_(b)CH₃), 1.19-1.06 (m, 1H, CH₂CH_(a)H_(b)CH₃), 0.78(app-t, J=7.0, 3H CH₂CH₂CH₃). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ215.9 (C═S), 164.7 (C₁₃), 160.5 (C₁₆), 141.6 (C₉), 140.4 (SO₂Ph-ipso-C),135.4 (C₄), 133.3 (SO₂Ph-p-C), 129.7 (C₇), 129.2 (SO₂Ph-m-C), 126.5(SO₂Ph-o-C), 125.9 (C₆), 123.6 (C₅), 117.6 (C₈), 83.7 (C₂), 74.6 (C₁₁),73.5 (C₁₅), 54.5 (C₃), 46.1 (C₁₂), 40.5 (CH₂CH₂CH₃), 28.5 (C₁₈), 19.7(C₁₇), 18.0 (CH₂CH₂CH₃), 14.3 (CH₂CH₂CH₃). FTIR (thin film) cm⁻¹: 1711(s), 1686 (s), 1477 (m), 1461 (m), 1447 (m), 1365 (s), 1167 (s), 732(m). HRMS (ESI) (m/z): calc'd for C₂₅H₂₅N₃NaO₄S₄ [M+Na]⁺: 582.0620,found 582.0646. TLC (40% ethyl acetate in hexanes), Rf: 0.18 (UV, CAM).

α-Epimer 67:

¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.79 (dd, J=1.0, 7.3, 2H, SO₂Ph-o-H),7.57 (d, J=8.0, 1H, C₈H), 7.53 (t, J=7.5, 1H, SO₂Ph-p-H), 7.40 (app-t,J=7.8, 2H, SO₂Ph-m-H), 7.27 (app-dt, J=1.4, 7.8, 1H, C₇H), 7.12 (app-dt,J=0.9, 7.6, 1H, C₆H), 7.06 (dd, J=0.8, 7.5, 1H, C₅H), 6.06 (s, 1H, C₂H),3.42 (d, J=15.7, 1H, C₁₂H_(a)), 2.97 (s, 3H, C₁₈H), 2.44 (d, J=15.7, 1H,C₁₂H_(b)), 1.95 (s, 3H, C₁₇H), 1.37-1.26 (m, 1H, CH₂CH_(a)H_(b)CH₃),1.26-1.14 (m, 1H, CH₂CH_(a)H_(b)CH₃), 0.97-0.83 (m, 2H, CH₂CH₂CH₃), 0.69(app-t, J=6.8, 3H, CH₂CH₂CH₃). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ216.8 (C═S), 164.3 (C₁₃), 161.4 (C₁₅), 139.3 (C₉), 138.9 (SO₂Ph-ipso-C),138.3 (C₄), 133.8 (SO₂Ph-p-C), 129.3 (SO₂Ph-m-C), 129.3 (C₇), 127.7(SO₂Ph-o-C), 126.3 (C₆), 124.4 (C₅), 118.3 (C₈), 84.5 (C₂), 74.8 (C₁₁),74.1 (C₁₅), 55.0 (C₃), 42.4 (C₁₂), 40.0 (CH₂CH₂CH₃), 28.6 (C₁₈), 21.0(C₁₇), 18.3 (CH₂CH₂CH₃), 14.2 (CH₂CH₂CH₃). FTIR (thin film) cm⁻¹: 1712(s), 1691 (s), 1476 (m), 1461 (m), 1447 (m), 1368 (s), 1333 (s), 1172(s), 727 (w). HRMS (ESI) (m/z): calc'd for C₂₅H₂₅N₃NaO₄S₄ [M+Na]⁺:582.0620, found 582.0636. TLC (40% ethyl acetate in hexanes), Rf: 0.50(UV, CAM).

β-C3-Propyl Epidithiodiketopiperazine 61:

Ethanolamine (500 μL) was added via syringe to a solution ofdithiepanethione 65 (13.3 mg, 23.8 μmol, 1 equiv) in acetone (500 L) at23° C. After 15 min, the reaction mixture was diluted withdichloromethane (30 mL) and aqueous hydrochloric acid solution (2 N, 30mL). The organic layer was collected, and the aqueous layer wasextracted with dichloromethane (2×2 mL). A solution of potassiumtriiodide in pyridine (2.5% w/v) was added dropwise to the combinedorganic layers until a persistent yellow color was observed. Theresulting mixture was washed with aqueous hydrochloric acid (2 N, 30 mL)and the layers were separated. The aqueous layer was extracted withdichloromethane (2×5 mL), and the combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: 1% acetone in dichloromethane) toafford epidithiodiketopiperazine 61 (8.6 mg, 70%) as a clear film. ¹HNMR (500 MHz, CDCl₃, 20° C.): δ 7.80 (d, J=7.0, 2H, SO₂Ph-o-H), 7.53 (t,J=7.0, 1H, SO₂Ph-p-H), 7.46-7.37 (m, 1H, C₈H), 7.46-7.37 (m, 2H,SO₂Ph-in-H), 7.29 (app-dt, J=1.1, 7.7, 1H, C₇H), 7.16 (app-t, J=7.6, 1H,C₆H), 7.12 (d, J=7.6, 1H, C₅H), 6.09 (s, 1H, C₂H), 3.19 (d, J=15.2, 1H,C₁₂H_(a)), 2.98 (s, 3H, C₁₈H), 2.57 (d, J=15.2, 1H, C₁₂H_(b)), 1.87 (s,3H, C₁₇H), 1.43-1.30 (m, 1H, CH₂CH_(a)H_(b)CH₃), 1.22-1.04 (m, 1H,CH₂CH_(a)H_(b)CH₃), 1.22-1.04 (m, 2H, CH₂CH₂CH₃), 0.77-0.68 (m, 3H,CH₂CH₂CH₃). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 165.9 (C₁₃), 161.6(C₁₆), 141.1 (C₉), 139.8 (SO₂Ph-ipso-C), 137.6 (C₄), 133.4 (SO₂Ph-p-C),129.3 (C₇), 129.2 (SO₂Ph-m-C), 127.4 (SO₂Ph-o-C), 125.9 (C₆), 123.6(C₅), 118.4 (C₈), 83.7 (C₂), 73.7 (C₁₁), 73.5 (C₁₅), 55.9 (C₃), 41.8(C₁₂), 40.0 (CH₂CH₂CH₃), 27.7 (C₁₈), 18.3 (CH₂CH₂CH₃), 18.0 (C₁₇), 14.3(CH₂CH₂CH₃). FTIR (thin film) cm⁻¹: 1713 (s), 1688 (s), 1478 (m), 1460(m), 1447 (m), 1341 (s), 1172 (s), 719 (w). HRMS (ESI) (m/z): calc'd forC₂₄H₂₅N₃NaO₄S₃ [M+Na]⁺: 538.0899, found 538.0923. TLC (1% acetone indichloromethane), Rf: 0.21 (UV, CAM).

α-C3-Propyl Epidithiodiketopiperazine 63:

Ethanolamine (500 μL) was added via syringe to a solution ofdithiepanethione 67 (13.3 mg, 23.8 μmol, 1 equiv) in acetone (500 μL) at23° C. After 15 min, the reaction mixture was diluted withdichloromethane (30 mL) and aqueous hydrochloric acid solution (2 N, 30mL). The organic layer was collected, and the aqueous layer wasextracted with dichloromethane (2×5 mL). A solution of potassiumtriiodide in pyridine (2.5% w/v) was added dropwise to the combinedorganic layers until a persistent yellow color was observed. Theresulting mixture was washed with aqueous hydrochloric acid (2 N, 30 mL)and the layers were separated. The aqueous layer was extracted withdichloromethane (2×5 mL), and the combined organic layers were driedover anhydrous sodium sulfate, were filtered, and were concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (eluent: 30% ethyl acetate indichloromethane) to afford epidithiodiketopiperazine 63 (9.6 mg, 78%) asa clear film. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.83 (dd, J=0.8, 8.2,2H, SO₂Ph-o-H), 7.53 (t, J=7.4, 1H, SO₂Ph-p-H), 7.51 (d, J=7.9, 1H,C₈H), 7.40 (app-t, J=8.1, 2H, SO₂Ph-m-H), 7.28-7.19 (m, 1H, C₇H),7.13-7.05 (m, 1H, C₆H), 7.13-7.05 (m, 1H, C₅H), 6.14 (s, 1H, C₂H), 3.57(d, J=14.9, 1H, C₁₂H_(a)), 2.89 (s, 3H, C₁₈H), 2.37 (d, J=14.9, 1H,C₁₂H_(b)), 1.93 (s, 3H, C₁₇H), 1.38-1.14 (m, 2H, CH₂CH₂CH₃), 1.00-0.85(m, 2H, CH₂CH₂CH₃), 0.70 (app-t, J=7.2, 3H, CH₂CH₂CH₃). ¹³C NMR (125.8MHz, CDCl₃, 20° C.): δ 165.7 (C₁₃), 162.9 (C₁₆), 139.3 (C₉), 139.1(SO₂Ph-ipso-C), 137.4 (C₄), 133.7 (SO₂Ph-p-C), 129.3 (SO₂Ph-m-C), 129.3(C₇), 127.7 (SO₂Ph-o-C), 126.1 (C₆), 124.5 (C₅), 118.1 (C₈), 84.3 (C₂),74.6 (C₁₁), 73.9 (C₁₅), 56.4 (C₃), 40.5 (C₁₂), 39.7 (CH₂CH₂CH₃), 27.5(C₁₈), 18.7 (C₁₇), 18.1 (CH₂CH₂CH₃), 14.2 (CH₂CH₂CH₃). FTIR (thin film)cm⁻¹: 1694 (s), 1447 (m), 1366 (s), 1331 (m), 1172 (s), 722 (w). HRMS(ESI) (m/z): calc'd for C₂₄H₂₅N₃NaO₄S₃ [M+Na]⁺: 538.0899, found538.0920. TLC (30% ethyl acetate in hexanes), Rf: 0.21 (UV, CAM).

Dimeric Bis(Triphenylmethanetrisulflde) 19: Anhydrous hydrazine (0.8 μL,25 μmol, 5.00 equiv) was added via syringe to a solution ofdiaminodithioisobutyrate (+)-S5 (6.6 mg, 5.0 μmol, 1 equiv) intetrahydrofuran (2 mL) at 0° C. After 18 min, triethylamine (17.5 μL,126 μmol, 25.0 equiv) and solid chloro(triphenylmethyl)disulfane (17.2mg, 50.3 μmol, 10.0 equiv) were sequentially added to the reactionmixture under an inert atmosphere. After 13 min, saturated aqueousammonium chloride (3 mL) was added to the reaction mixture. The solutionwas then poured into a separatory funnel containing saturated aqueousammonium chloride (10 mL) and dichloromethane (15 mL). The aqueous layerwas extracted with dichloromethane (2×5 mL), and the combined organiclayers were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (eluent: 35% ethyl acetate inhexanes) to afford dimeric bis(triphenylmethanetrisulfide) (+)-19 (7.4mg, 82%) as a slightly off-white solid. ¹H NMR (500 MHz, CDCl₃, 20° C.):δ 8.04 (d, J=7.5, 4H, SO₂Ph-o-H), 7.64 (t, J=7.5, 2H, SO₂Ph-p-H), 7.52(app-t, J=7.9, 4H, SO₂Ph-m-H), 7.22-7.12 (m, 2H, C₈H), 7.22-7.12 (m,18H, C(C₆H₅)), 6.99-6.90 (m, 12H, C(C₆H₅)₃), 6.80 (s, 2H, C₂H), 6.65(br-s, 2H, C₅H), 6.57 (app-t, J=8.1, 2H, C₇H), 6.08 (app-t, J=7.0, 2H,C₆H), 4.43 (d, J=11.9, 2H, C₁₇H_(a)), 4.23 (d, J=11.7, 2H, C₁₇H_(b)),3.31 (d, J=14.5, 2H, C₁₂H_(a)), 2.92 (d, J=14.4, 2H, C₁₂H_(b)), 2.71 (s,6H, C₁₈H), 2.54 (app-sp, J=7.1, 2H, CH_(isobutyrate)), 1.79 (s, 6H,CH_(3acetate)), 1.11 (d, J=7.0, 6H, CH_(3isobutyrate)), 1.08 (d, J=7.1,6H, CH_(3isobutyrate)). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 174.1(C═O_(isobutyrate)), 170.1 (C═O_(acetate)), 164.1 (C₁₃), 161.2 (C₁₆),143.3 (C(C₆H₅)₃), 142.8 (C₉), 140.5 (SO₂Ph-ipso-C), 133.4 (SO₂Ph-p-C),130.7 (C₄), 130.5 (C(C₆H₅)₃), 129.6 (SO₂Ph-m-C), 129.4 (C₇), 128.0(C(C₆H₅)₃), 127.3 (C(C₆H₅)₃), 127.3 (SO₂Ph-o-C), 124.0 (C₅), 123.8 (C₆),112.8 (C₈), 86.2 (C₁₅), 80.9 (C₂), 75.2 (C₁₁), 73.3 (C(C₆H₅)₃), 64.7(C₁₇), 60.7 (C₃), 42.8 (C₁₂), 33.6 (CH_(isobutyrate)), 28.7 (C₁₈), 21.4(CH_(3acetate)), 18.8 (CH_(3isobutyrate)). FTIR (thin film) cm⁻¹: 1749(s), 1708 (s), 1480 (m), 1462 (m), 1447 (m), 1380 (s), 1220 (m), 1173(s), 729 (m), 699 (m). HRMS (ESI) (m/z): calc'd for C₉₂H₈₈N₇O₁₆S₈[M+NH₄]⁺: 1802.4048, found 1802.4073. [α]_(D) ²⁴: +287 (c 0.35, CHCl₃).TLC (35% ethyl acetate in hexanes), Rf: 0.23 (UV, CAM).

Among other things, the present invention recognizes that it isparticularly challenging to prepare ETP or thiodiketopiperazinecompounds wherein one of R⁶ and R^(6′) is —OR or —OSi(R)₃. In someembodiments, the present provides new methods for preparing a providedcompounds, wherein one of R⁶ and R^(6′) is —OR or —OSi(R)₃. In someembodiments, such a compound, for example, a compound having thestructure of formula I-c or I-d, is depicted below:

Exemplary C12-Hydroxylated Epipolythiodiketopiperazines

In some embodiments, a provided new method comprises an intermolecularFriedel-Crafts reaction of a silyl-tethered indole moiety. In someembodiments, the present invention provides scalable methods forerythro-β-hydroxytryptophan amino acid synthesis. In some embodiments,the present invention provide new mercaptan reagent for synthesis of,for example, epipolythiodiketopiperazine (ETP) or thiodiketopiperazinescompounds, and/or derivatives and analogs thereof. In some embodiments,a provided mercaptan reagent can be unraveled under a mild conditionthat does not disrupt an N-protecting group of an indolyl moiety. Insome embodiments, a provided mercaptan reagent can be unraveled under amild condition that does not disrupt an N-protecting group of an indolylmoiety, wherein the protecting group is —S(O)₂R. In some embodiments, aprovided mercaptan reagent can be unraveled under a mild condition thatdoes not disrupt an N-protecting group of an indolyl moiety, wherein theprotecting group is —S(O)₂Ph. In some embodiments, the present inventionprovides a method of permanganate-mediated stereoinvertive hydroxylationof the α-stereocenters of diketopiperazines. In some embodiments, thepresent invention provides a method of direct triketopiperazinesynthesis from cyclo-dipeptides. Non-limiting examples are describedherein.

In some embodiments, the present invention provides a general method forpreparing epidithiodiketopiperazine alkaloids, for example, a providedcompound of formulae I-a, I-b, I-c and I-d, by using β-hydroxytryptophanin lieu of tryptophan as our feedstock material.

An exemplary retrosynthetic analysis of (+)-bionectins A (E2-1) and C(E2-2) is outlined in Scheme E2-1.

In some embodiments, an exemplary synthesis of (+)-bionectins A and Ccommenced with the development of a concise and scalable route toerythro-β-hydroxytryptophan (Scheme E2-2). While several methods havebeen reported for the synthesis of the threo diastereomer ((a) H.Sugiyama, T. Shioiri and F. Yokokawa, Tetrahedron Lett., 2002, 43, 3489;(b) S.-J. Wen, H.-W. Zhang and Z.-J. Yao, Tetrahedron Lett., 2002, 43,5291; (c) K. S. Feldman and A. G. Karatjas, Org. Lett., 2004, 6, 2489;(d) S.-J. Wen and Z.-J. Yao, Org. Lett., 2004, 6, 2721; (e) D. Crich andA. Banerjee, J. Org. Chem., 2006, 71, 7106; (f) D. B. Hansen, A. S.Lewis, S. J. Gavalas and M. M. Joullié, Tetrahedron: Asymmetry, 2006,17, 15; (g) J. Patel, G. Clavé, P.-Y. Renard and X. Franck, Angew.Chem., Int. Ed., 2008, 47, 4224), there was no scalable available methodto the desired tryptophan derivative. After extensive optimization itwas surprisingly found that titanium (IV)-mediated anti-aldol reaction(A. Solladiè-Cavallo and J. L. Koessler, J. Org. Chem., 1994, 59, 3240;(b) A. Solladiè-Cavallo and T. Nsenda, Tetrahedron Lett., 1998, 39,2191; (c) A. Teniou and H. Alliouche, Asian J. Chem., 2006, 18, 2487) toindole-3-carboxaldehyde E2-10 and (−)-pinanone-derived ethyliminogylcinate E2-11 (T. Oguri, N. Kawai, T. Shioiri and S.-i. Yamada,Chem. Pharm. Bull., 1978, 26, 803) efficiently afforded the aldoladducts, in some embodiments, in 81% yield on greater than 40 gramscale. Subsequent hydrolysis of the Schiff base then afforded greaterthan 20 grams of β-hydroxy-α-amino ester E2-13 in 94% ee along withrecovery of the chiral auxiliary. The absolute and relativestereochemistry of the material were verified through single crystalX-ray diffraction analysis of its 3,5-dinitrobenzamide derivative E2-14.

With a rapid and scalable route to erythro-β-hydroxytryptophanavailable, we proceeded to the synthesis of the desired tetracycleE2-16. Dipeptide formation with N-Boc-sarcosine followed by unveiling ofthe amine and intramolecular cyclization with AcOH and morpholine intert-butanol afforded diketopiperazine E2-15 in 97% yield. Exposure ofdiketopiperazine E2-15 to excess bromine in MeCN at 0° C., andsubsequent addition of anisole led to a diastereoselectivehalocyclization with concomitant loss of the silyl ether. In someembodiments, in addition to preventing undesired ring-halogenation,quenching of excess bromine with anisole resulted in the in situformation of hydrobromic acid, which was responsible for the desiredremoval of the silyl ether function. Under these optimized conditions,tetracyclic bromide E2-16 could be accessed in decagram quantities in94% yield (9:1 dr, endo: exo) favoring the desired diastereomer. In someembodiments, we found that the C12 hydroxyl group favors the formationof the desired endo-cyclization product independent of the ancillaryamino acid substituent at the C15 center.

Using the tetracyclic bromide, we tried to implement an intermolecularFriedel-Crafts indolylation at the C3 position in a manner akin togliocladin synthesis; however, without the intention to be limited bytheory, due to the inductive effects of the C12-hydroxyl group (SchemeE2-3), we found the C3-bromide proved recalcitrant toward ionization.Under more forcing conditions, C3-carbocation derivatives E2-18 could beformed, but their instability required rapid trapping, a feat in someembodiments hindered by the additional substitution at C12. Applicationof the conditions for intermolecular Friedel-Crafts reaction ((a) J. Kimand M. Movassaghi, J. Am. Chem. Soc., 2011, 133, 14940; (b) N. Boyer andM. Movassaghi, Chem. Sci., 2012, 3, 1798) resulted in regioisomeric anddiastereomeric products (Scheme E2-3). In some embodiments, the geometryof the tricyclic substructure was insufficient in overcoming the stericpressures imposed by C12-hydroxylation, resulting in 10% undesiredbyproducts consistent with indole addition from the concave face.

After examining a variety of strategies, it was surprisingly found thatan intramolecular delivery of the indole fragment provided the desiredproduct efficiently. Silylation of tetracyclic alcohol withchlorodimethyl(N-Boc-2-indole)silane (E2-20, S. E. Denmark and J. D.Baird, Org. Lett., 2004, 6, 3649) provided the desired silyl-tetheredindole adduct E2-21 in 74% yield (Scheme E2-4). Surprisingly, asilver-mediated intramolecular Friedel-Crafts reaction proceededsmoothly in nitroethane at 0° C. to afford the C3-(3′-indolyl)-salicylicproduct E2-22 in 68% yield. The structure of a diethyl silyl variantE2-23 was confirmed through X-ray analysis (Scheme E2-4). The desiredC3-indolylated tetracycle E2-24 was accessed in 58% yield by treatmentof salicylic product E2-22 with aqueous hydrochloric acid.

The key indolylated intermediate E2-24 was subsequentlybis(tert-butoxycarbonyl) protected in 92% yield using Boc₂O and DMAP inanticipation of our C—H hydroxylation chemistry. In some embodiments,the hydroxylation proceeded effectively in the presence of lesselectron-rich substructures (Scheme E2-4). In some embodiments, theoxidation was more efficient with the tert-butoxycarbonyl group on N1′on this system. Surprisingly, rather than providing the expectedstereoretentive dihydroxylation product (Scheme E2-1), in someembodiments, oxidation of the tetracycle with excessbis(pyridine)silver(I) permanganate in dichloromethane affordedtriketopiperazine 25 in 45% yield as a single diastereomer, representingan average of 77% yield per oxidation event. Direct access to atriketopiperazine motif is a highly enabling transformation, and can beused to preparation C-15 derivatives, for example, as described in J. E.DeLorbe, D. Home, R. Jove, S. M. Mennen, S. Nam, F.-L. Zhang and L. E.Overman, J. Am. Chem. Soc., 2013, 135, 4117.

The C15 carbonyl group of triketopiperazine E2-25 was reduced in ahighly diastereoselective fashion using sodium borohydride in methanolat −20° C. to afford the desired diol E2-27 in 75% yield (Scheme E2-4).The relative stereochemistries of the C11 and C15 alcohols were thenverified by peracetylation of a C12-acetylated diol derivative followedby single crystal X-ray diffraction analysis of the resultant triacetateE2-28. Surprisingly, the C11 stereochemistry is consistent withhydroxylation with inversion of the originating C—H stereochemistry, anevent unprecedented in other oxidations of diketopiperazines withoutC12-hydroxylation ((a) J. Kim, J. A. Ashenhurst and M. Movassaghi,Science, 2009, 324, 238; (b) J. Kim and M. Movassaghi, J. Am. Chem.Soc., 2010, 132, 14376. (c) N. Boyer and M. Movassaghi, Chem. Sci.,2012, 3, 1798). While not wishing to be limited by theory, Applicantnotes that it is likely that the captodatively-stabilized radicalresulting from permanganate-mediated C—H abstraction is stericallyshielded by the C12-tert-butoxycarbonate group, preventing thesubsequent hydroxylation step through a rapid rebound mechanism (K. A.Gardner and J. M. Mayer, Science, 1995, 269, 1849; (b) T. Strassner andK. N. Houk, J. Am. Chem. Soc., 2000, 122, 7821); reaction of apermanganate molecule with the persistent, stereochemically labilecarbon-centered radical on the opposite face of the diketopiperazinewould afford the oxidation product.

In some embodiments, recognizing the C11 and C15 alcohols to berecalcitrant toward ionization by virtue of their proximity to aninductively withdrawing carbonate and their location on a secondarycarbon, respectively, the hydroxyl groups were activated for ionizationby acylation with pivaloyl chloride and DMAP in dichloromethane toprovide dipivaloate E2-27 in 83% yield. Constraining our search tofunctional groups capable of withstanding conditions for thephotoinduced reductive removal of a benzenesulfonyl group, we initiallyevaluated the use of thioacid and alkyl mercaptan nucleophiles. Whilethioacids resulted in categorically low levels of diastereoselection forthe nucleophilic addition, alkyl mercaptans proved highlydiastereoselective on this substrate in affording their bisthioetheradduct. However, known thioether reagents such as 2-cyanoethyl and2-trimethylsilylethyl mercaptans required intolerably harsh conditionsfor their conversion to the necessary thiols.

TABLE E2-1 Stereoselective sulfidation of diketopiperazines.

Bissulfide yield^(a) ETP yield

70%

65%

78%

60%

75%

57% Conditions: (a) E2-29, TFA, MeCN, 23° C.; (b) E2-30, TFA, MeCN, 23°C.; (c) pyrrolidine, O₂, MeCN, 23° C. ^(a)Isolated as a singlediastereomer. R_(Me) = CH₂CH₂(CO)Me, R_(Ph) = CH₂CH₂(CO)Ph.

In some embodiments, the present invention provides new sulphidesurrogates, and new methods for sulfidation. We generated4-mercaptobutan-2-one (E2-29) (N. C. Ross and R. Levine, J. Org. Chem.,1964, 29, 2346) by addition of hydrogen sulphide to methyl vinyl ketoneand 3-mercaptopropiophenone (E2-30) by addition of thioacetic acid to3-chloropropiophenone followed by hydrolysis. Exposure of severaldiketopiperazine-derived bishemiaminals to trifluoroacetic acid inacetonitrile gratifyingly resulted in diastereoselective cis-thioetheradducts (Table E2-1). While the additions were highly diastereoselectiveusing either of our thiol reagents on our bisproline substrate, in someembodiments, mercaptan E2-30 afforded superior diastereoselectivities tomercaptan E2-29 on other substrates including the diol precursor tobisthioether E2-33. In some embodiments, bisthioether adducts ofmethylketone-based mercaptan E2-29 underwent pyrrolidine-catalyzedsulfide-cleavage at a faster rate and was better suited for use withmore sensitive compounds such as substrate E2-27.

The bisthioethers generated using this new method could be converted tothe corresponding epidithiodiketopiperazine under exceedingly mildconditions. Addition of pyrrolidine to a solution of the adducts inacetonitrile under an atmosphere of oxygen resulted in the directconversion of substrates E2-31-E2-33 to their corresponding disulphides(Table E2-1). In the event that a dithiol cannot be oxidized readilywith molecular oxygen to the disulphide in order to drive theβ-addition/elimination equilibration process toward product formation, asacrificial thiol can be added to the reaction mixture to effect atransthioetherification.

Indeed, application of the provided new methodology for sulfidation ofdiketopiperazines proved critical in the synthesis of (+)-bionectins Aand C. Treatment of a solution of dipivaloate E2-27 and ketomercaptanreagent E2-29 with trifluoroacetic acid in nitromethane at 23° C.yielded a diastereomeric mixture of bisthioethers E2-36 in 80% yield and3:1 dr with concomitant removal of the tert-butoxycarbonyl groups at theN1′ amine and C12 alcohol. While not wishing to be limited by theory,Applicant notes that ionization at C11 did not occur in acetonitrilelikely due to the inductive effects of the C12-hydroxy group. The majordiastereomer possessed the desired C11, C15-stereochemistry and could beisolated in 56% yield upon photoinduced electron transfer-mediatedremoval of the benzenesulfonyl group (T. Hamada, A. Nishida and O.Yonemitsu, J. Am. Chem. Soc., 1986, 108, 140). The bisthioethers werethen removed with a mild enamine-mediated transthioetherificationprotocol employing pyrrolidine and ethanethiol in THF. The use of asacrificial thiol was surprisingly found to be optimal in the unveilingof the thiols; exposure to an atmosphere of oxygen was insufficient inoxidizing the dithiol to a disulphide. Without the intention to belimited by theory, Applicant notes that the C15 thiol may prefer anequatorial disposition in its ground state and that conformation may notbe as conducive to oxidation by molecular oxygen. Mild oxidation withKI₃ in pyridine then afforded the target compound (+)-bionectin A (E2-1)in 81% yield.

We treated the synthetic sample of (+)-E2-1 with p-nitrobenzoyl chlorideand DMAP in CH₂Cl₂ at 0° C. to afford (+)-bionectin A-p-nitrobenzoate(E2-38) in 98% yield. Single crystal X-ray diffraction analysis of thisC12-p-nitrobenzoate derivative confirmed its structure. Reductivemethylation of (+)-bionectin A (E2-1) with sodium borohydride and MeI inpyridine and methanol afforded (+)-bionectin C (E2-2) in 97% yield (H.Poisel and U. Schmidt, Chem. Ber., 1971, 104, 1714).

General Procedures.

All reactions were performed in oven-dried or flame-dried round-bottomflasks. The flasks were fitted with rubber septa and reactions wereconducted under a positive pressure of argon. Cannulae or gas-tightsyringes with stainless steel needles were used to transfer air- ormoisture-sensitive liquids. Where necessary (so noted), solutions weredeoxygenated by sparging with argon for a minimum of 10 min. Flashcolumn chromatography was performed as described by Still et al. usinggranular silica gel (60-Å pore size, 40-63 μm, 4-6% H₂O content,Zeochem) (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,2923). Analytical thin layer chromatography (TLC) was performed usingglass plates pre-coated with 0.25 mm 230-400 mesh silica gel impregnatedwith a fluorescent indicator (254 nm). TLC plates were visualized byexposure to short wave ultraviolet light (254 nm) and an aqueoussolution of ceric ammonium molybdate (CAM) followed by heating on a hotplate (˜250° C.). Organic solutions were concentrated at 29-30° C. onrotary evaporators capable of achieving a minimum pressure of ˜2 torr.The benzenesulfonyl photodeprotection was accomplished by irradiation ina Rayonet RMR-200 photochemical reactor (Southern New EnglandUltraviolet Company, Branford, Conn., USA) equipped with 16 lamps(RPR-3500, 24 W, λ_(max)=350 nm, bandwidth 20 nm).

Materials.

Commercial reagents and solvents were used as received with thefollowing exceptions: dichloromethane, acetonitrile, tetrahydrofuran,methanol, pyridine, toluene, and triethylamine were purchased from J.T.Baker (Cycletainer™) and were purified by the method of Grubbs et al.under positive argon pressure (Pangborn, A. B.; Giardello, M. A.;Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15,1518). Nitromethane and nitroethane (from Sigma-Aldrich) were purifiedby fractional distillation over calcium hydride and were stored overLinde 4 Å molecular sieves in Schlenk flasks sealed with septa andteflon tape under argon atmosphere (Armarego, W. L. F.; Chai, C. L. L.Purification of Laboratory Chemicals, 5^(th) ed.; Butterworth-Heinemann:London, 2003). Titanium (IV) ethoxide (99.99%-Ti) PURATREM and brominewere purchased from Strem Chemicals, Inc.; N-Boc-L-sarcosine,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride,N-hydroxybenzotriazole, tert-butyldimethylsilyltrifluoromethanesulfonate, trifluoroacetic acid,4-(dimethylamino)pyridine, silver nitrate were purchased fromChem-Impex; 1,4-dimethoxynaphthalene and iodomethane were purchased fromAlfa Aesar; di-tert-butyl dicarbonate was purchased from OakwoodProducts, Inc.; 2,6-di-tert-butyl-4-methylpyridine (DTBMP) was purchasedfrom OChem Incorporation. All other solvents and chemicals werepurchased from Sigma-Aldrich. 1,4-Dimethoxynaphthalene was purified bycrystallization from absolute ethanol.

Instrumentation.

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded with aBruker AVANCE-600 NMR spectrometer (with a Magnex Scientificsuperconducting actively-shielded magnet) or with a Varian inverse probe500 INOVA spectrometer, are reported in parts per million on the δscale, and are referenced from the residual protium in the NMR solvent(CDCl₃: δ 7.26 (CHCl₃) or DMSO-d₆: δ 2.50 (DMSO-d₅)) (Fulmer, G. R.;Miller, A. J. M.; Sherden, N. H.; Gottlieb, H. E.; Nudelman, A.; Stoltz,B. M.; Bercaw, J. E.; Goldberg, K. I. Organometallics 2010, 29, 2176).Data are reported as follows: chemical shift [multiplicity (br=broad,s=singlet, d=doublet, t=triplet, sp=septet, m=multiplet), couplingconstant(s) in Hertz, integration, assignment]. Carbon-13 nuclearmagnetic resonance (¹³C NMR) spectra were recorded with a BrukerAVANCE-600 NMR Spectrometer (with a Magnex Scientific superconductingactively-shielded magnet) or a Bruker AVANCE-400 NMR Spectrometer (witha Magnex Scientific superconducting magnet) or with a Varian 500 INOVAspectrometer, are reported in parts per million on the δ scale, and arereferenced from the carbon resonances of the solvent (CDCl₃: δ 77.23 orDMSO-d₆: δ 39.52). Data are reported as follows: chemical shift(multiplicity, coupling constant(s) in Hertz, assignment). Infrared data(IR) were obtained with a Perkin-Elmer 2000 FTIR and are reported asfollows: frequency of absorption (cm⁻¹), intensity of absorption(s=strong, m=medium, w=weak, br=broad). Optical rotations were measuredon a Jasco-1010 polarimeter with a sodium lamp and are reported asfollows: [α]_(λ) ^(T ° C.) (c=g/100 mL, solvent). We are grateful to Dr.Li Li and Deborah Bass for obtaining the mass spectrometric data at theDepartment of Chemistry's Instrumentation Facility, MassachusettsInstitute of Technology. High resolution mass spectra (HRMS) wererecorded on a Bruker Daltonics APEXIV 4.7 Tesla FT-ICR-MS using anelectrospray (ESI) ionization source.

Positional Numbering System.

At least three numbering systems for dimeric diketopiperazine alkaloidsexist in the literature ((a) Von Hauser, D.; Weber, H. P.; Sigg, H. P.Helv. Chim. Acta 1970, 53, 1061. (b) Barrow, C. J.; Cai, P.; Snyder, J.K.; Sedlock, D. M.; Sun, H. H.; Cooper, R. J. Org. Chem. 1993, 58, 6016.(c) Springer, J. P.; Büchi, G.; Kobbe, B.; Demain, A. L.; Clardy, J.Tetrahedron Lett. 1977, 28, 2403. (d) Zheng, C.-J.; Kim, C.-J.; Bae, K.S.; Kim, Y.-H.; Kim, W.-G J. Nat. Prod. 2006, 69, 1816. (e) DeLorbe, J.E.; Jabri, S. Y.; Mennen, S. M.; Overman, L. E.; Zhang, F.-L. J. Am.Chem. Soc. 2011, 133, 6549). In assigning the ¹H and ¹³C NMR data of allintermediates en route to our total syntheses of (+)-bionectins A (E2-1)and C (E2-2), we wished to employ a uniform numbering scheme. For easeof direct comparison, particularly between early intermediates,non-thiolated diketopiperazines, and advanced compounds, the numberingsystem used by Barrow for (+)-WIN-64821 (using positional numbers 1-21)is optimal and used throughout this report. In key instances, theproducts are accompanied by the numbering system as shown below.

12-Hydroxytryptophan Alcohol E2-12:

A solution of chlorotitanium (IV) triethoxide (22.5 g, 103 mmol, 1.05equiv, Holoway, H. Chem. Ind. 1962, 3, 214) in dichloromethane (69 mL)was added via cannula to a solution of ethyl2-((1S,2S,5S)-2-hydroxypinan-3-imino)glycinate (E2-11, 24.8 g, 97.8mmol, 1 equiv, (a) Oguri, T.; Kaway, N.; Yamada, S. Chem. Pharm. Bull.1978, 26, 803. (b) Solladiè-Cavallo, A.; Simon, M. C. Tetrahedron Lett.1989, 30, 6011. (c) Solladiè-Cavallo, A.; Simon-Wermeister, M. C.;Schwarz, J. Organometallics 1993, 12, 3743) in dichloromethane (300 mL)at 0° C. A fine powder of 1-(phenylsulfonyl)-1H-indole-3-carbaldehyde(E2-10, 29.3 g, 103 mmol, 1.05 equiv, Wenkert, E.; Moeller, P. D. R.;Piettre, S. R. J. Am. Chem. Soc. 1988, 110, 7188) was then added as asolid to the reaction mixture. Triethylamine (27.3 mL, 196 mmol, 2.00equiv) was subsequently added dropwise via syringe and the reactionmixture was stirred at 0° C. After 21 h, brine (1 L) at 0° C. was addedto the reaction mixture and the resulting bilayer suspension wasfiltered through Celite. The organic layer was separated, and theaqueous layer was extracted with dichloromethane (2×300 mL). Thecombined organic layers were dried over anhydrous sodium sulfate, werefiltered, and were concentrated under reduced pressure. The resultingorange foam was purified by flash column chromatography on silica gel(eluent: gradient, 30→50% ethyl acetate in hexanes) to provide aninseparable mixture of diastereomeric aldol products (42.5 g, 80.6%) asa yellow foam. In some embodiments, the aldol products were highly proneto degradation through a retro-aldol pathway; thus, the mixture ofdiastereomers was quickly isolated and immediately used in thesubsequent reaction. Structural assignments were made using additionalinformation from gCOSY, HSQC, and gHMBC experiments.

¹H NMR (600 MHz, CDCl₃, 20° C.; only the peaks corresponding to themajor diastereomer are tabulated): δ 7.96 (d, J=8.3, 1H, C₅H), 7.86 (d,J=8.6, 2H, SO₂Ph-o-H), 7.71 (s, 1H, C₂H), 7.67 (d, J=7.8, 1H, C₅H), 7.49(t, J=7.6, 1H, SO₂Ph-p-H), 7.39 (app-t, J=8.1, 2H, SO₂Ph-m-H), 7.29(app-t, J=7.3, 1H, C₇H), 7.23 (app-t, J=7.2, 1H, C₆H), 5.49 (d, J=6.9,1H, C₁₂H), 4.46 (d, J=6.9, 1H, C₁₁H), 4.11-3.99 (m, 2H, CO₂CH₂CH₃), 3.90(br-s, 1H, C₁₂OH), 2.42 (dd, J=2.0, 17.7, 1H, C₁₆H_(a)), 2.19 (dd,J=2.7, 18.0, 1H, C₁₆H_(b)), 2.11-2.05 (m, 1H, C₁₈H_(a)), 2.00 (br-s, 1H,C₂₀OH), 1.89 (app-t, J=5.8, 1H, C₁₉H), 1.87-1.83 (m, 1H, C₁₇H), 1.42 (s,3H, C₂₄H), 1.23 (s, 3H, C_(22/23)H), 1.06 (app-t, J=7.4, 3H, CO₂CH₂CH₃),1.02 (d, J=4.7, 1H, C₁₈H_(b)), 0.80 (s, 3H, C_(22/23)H). ¹³C NMR (150MHz, CDCl₃, 20° C.): δ 180.6 (C₁₅), 169.5 (C₃), 138.0 (SO₂Ph-ipso-C),134.5 (C₉), 133.6 (SO₂Ph-p-C), 129.9 (C₄), 129.2 (SO₂Ph-m-C), 126.7(SO₂Ph-o-C), 124.9 (C₇), 124.7 (C₂), 123.1 (C₆), 122.3 (C₃), 120.2 (C₅),113.5 (C₈), 76.8 (C₂₀), 67.7 (C₁₂), 67.4 (C₁₁), 61.1 (CO₂CH₂CH₃), 50.3(C₁₉), 38.5 (C₂₁), 38.2 (C₁₇), 33.8 (C₁₆), 28.1 (C₂₄), 27.8 (C₁₈), 27.1(C22/23), 22.6 (C_(22/23)), 13.7 (CO₂CH₂CH₃). FTIR (thin film) cm⁻¹:3422 (br-m), 2926 (s), 1734 (s), 1649 (m), 1557 (w), 1448 (s), 1373 (s),1273 (m), 1181 (s), 1126 (m), 1089 (m), 1022 (w), 978 (w), 920 (w), 751(m). HRMS (ESI) (m/z): calc'd for C₂₉H3₅N₂O₆S [M+H]⁺: 539.2210, found:539.2198. TLC (50% ethyl acetate in hexanes), Rf. 0.49 (UV, CAM, KMnO₄).

12-Hydroxytryptophan Silyl Ether (+)-E2-S1:

t-Butyldimethylsilyl trifluoromethanesulfonate (21.8 mL, 94.7 mmol, 1.20equiv) was added via syringe to a solution of 12-hydroxytryptophanalcohol E2-12 (42.5 g, 78.9 mmol, 1 equiv) and 2,6-lutidine (18.7 mL,161 mmol, 2.04 equiv) in dichloromethane (900 mL) at 0° C. After 2 h,saturated aqueous ammonium chloride solution (750 mL) was added to thereaction mixture and the resulting solution was allowed to warm to 23°C. After 10 min, the layers were separated and the aqueous layer wasfurther extracted with dichloromethane (2×200 mL). The combined organiclayers were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The resulting orange foam waspurified by flash column chromatography on silica gel (eluent: 20% ethylacetate in hexanes) to provide the 12-hydroxytryptophan silyl ether(+)-E2-S1 (37.1 g, 72.0%) as a yellow oil. Structural assignments weremade using additional information from gCOSY, HSQC, and gHMBCexperiments.

¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.97 (d, J=8.3, 1H, C₈H), 7.84 (d,J=7.4, 2H, SO₂Ph-o-H), 7.77 (d, J=7.8, 1H, C₅H), 7.52 (s, 1H, C₂H), 7.50(t, J=7.9, 1H, SO₂Ph-p-H), 7.39 (app-t, J=8.2, 2H, SO₂Ph-m-H), 7.30(app-t, J=7.3, 1H, C₇H), 7.24 (app-t, J=7.1, 1H, C₆H), 5.53 (d, J=8.5,1H, C₁₂H), 4.41 (d, J=8.6, 1H, C₁₁H), 4.23-4.14 (m, 2H, CO₂CH₂CH₃), 2.42(app-dt, J=2.5, 18.1, 1H, C₁₆H_(a)), 2.00 (dd, J=2.9, 18.1, 1H,C₁₆H_(b)), 1.93-1.90 (m, 1H, C₁₈H_(a)), 1.80 (app-t, J=5.8, 1H, C₁₉H),1.75-1.71 (m, 1H, C₁₇H), 1.42 (br-s, 1H, C₂₀OH), 1.31 (s, 3H, C₂₄H),1.06 (app-t, J=7.2, 3H, CO₂CH₂CH₃), 1.18 (s, 3H, C_(22/23)H), 0.82 (s,9H, Si(CH₃)₂C(CH₃)₃), 0.70 (d, J=5.2, 1H, C₁₅H_(b)), 0.69 (s, 3H,C_(22/23)H), 0.03 (s, 3H, Si(CH₃)₂C(CH₃)₃), −0.30 (s, 3H,Si(CH₃)₂C(CH₃)₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 180.2 (C₁₅), 170.0(C₁₃), 138.3 (SO₂Ph-ipso-C), 135.2 (C₉), 133.9 (SO₂Ph-p-C), 129.5 (C₄),129.4 (SO₂Ph-m-C), 126.8 (SO₂Ph-o-C), 125.2 (C₇), 124.6 (C₃), 124.0(C₂), 123.3 (C₆), 121.0 (C₅), 114.1 (C₈), 76.4 (C₂₀), 70.7 (C₁₁), 70.0(C₁₂), 61.1 (CO₂CH₂CH₃), 49.8 (C₁₉), 38.4 (C₂₁), 38.1 (C₁₇), 33.4 (C₁₆),28.3 (C₂₄), 27.8 (C₁₈), 27.3 (C_(22/23)), 25.7 (Si(CH₃)₂C(CH₃)₃)), 22.8(C_(22/23)), 18.1 (Si(CH₃)₂C(CH₃)₃), 14.4 (CO₂CH₂CH₃), −4.6(Si(CH₃)₂C(CH₃)₃), −5.3 (Si(CH₃)₂C(CH₃)₃). FTIR (thin film) cm⁻¹: 2929(s), 1735 (s), 1652 (m), 1559 (w), 1494 (w), 1448 (s), 1372 (s), 1259(m), 1179 (s), 1088 (s), 977 (w), 837 (m), 780 (m), 750 (m), 686 (m).HRMS (ESI) (m/z): calc'd for C₃₅H₄₉N₂O₆SSi [M+H]⁺: 653.3075, found:653.3063. [α]_(D) ²⁴: +3.1 (c=0.30, CHCl₃). TLC (33% ethyl acetate inhexanes), Rf: 0.42 (UV, CAM).

12-Hydroxytryptophan Amine (+)-E2-13:

Aqueous hydrogen chloride solution (2 N, 520 mL) was added to a solutionof 12-hydroxytryptophan silyl ether (+)-E2-S1 (37.1 g, 56.8 mmol, 1equiv) in tetrahydrofuran (520 mL) at 23° C. After 1.5 h, the mixturewas concentrated to remove the organic solvent. The resulting mixturewas extracted with ethyl acetate (3×500 mL). The combined organic layerswere dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The resulting orange foam waspurified by flash column chromatography on silica gel (eluent: gradient,20→30→100% ethyl acetate in hexanes) to provide 12-hydroxytryptophanamine (+)-E2-13 (23.0 g, 80.5%) as a yellow foam. Structural assignmentswere made using additional information from gCOSY, HSQC, and gHMBCexperiments. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 8.00 (d, J=8.3, 1H,C₈H), 7.83 (d, J=7.4, 2H, SO₂Ph-o-H), 7.64 (d, J=7.9, 1H, C₅H), 7.52 (t,J=7.4, 1H, SO₂Ph-p-H), 7.49 (s, 1H, C₂H), 7.41 (app-t, J=8.2, 2H,SO₂Ph-m-H), 7.32 (app-t, J=7.4, 1H, C₇H), 7.22 (app-t, J=7.3, 1H, C₆H),5.01 (d, J=6.5, 1H, C₁₂H), 4.10-3.98 (m, 2H, CO₂CH₂CH₃), 3.78 (d, J=6.5,1H, C₁₁H), 1.13 (app-t, J=7.1, 3H, CO₂CH₂CH₃), 0.80 (s, 9H,Si(CH₃)₂C(CH₃)₃), 0.00 (s, 3H, Si(CH₃)₂C(CH₃)₃), −0.33 (s, 3H,Si(CH₃)₂C(CH₃)₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 172.9 (C₁₃), 138.1(SO₂Ph-ipso-C), 135.2 (C₉), 134.0 (SO₂Ph-p-C), 129.4 (SO₂Ph-m-C), 129.0(C₄), 126.8 (SO₂Ph-o-C), 125.2 (C₇), 124.6 (C₂), 123.5 (C₆), 122.9 (C₃),121.1 (C₅), 114.0 (C₈), 72.0 (C₁₂), 61.1 (CO₂CH₂CH₃), 60.8 (C₁₁), 25.7(Si(CH₃)₂C(CH₃)₃), 18.2 (Si(CH₃)₂C(CH₃)₃), 14.1 (CO₂CH₂CH₃), −4.8(Si(CH₃)₂C(CH₃)₃), −5.3 (Si(CH₃)₂C(CH₃)₃). FTIR (thin film) cm⁻¹: 2931(s), 2858 (s), 1735 (s), 1560 (w), 1448 (m), 1372 (s), 1258 (m), 1180(s), 1088 (m), 1023 (w), 976 (w), 838 (s), 751 (s), 686 (m). HRMS (ESI)(m/z): calc'd for C₂₅H₃₅N₂O₅SSi [M+H]⁺: 503.2030, found: 503.2016.[α]_(D) ²⁴: +31 (c=0.45, CHCl₃). TLC (50% ethyl acetate in hexanes), Rf:0.47 (UV, CAM, KMnO₄).

12-Hydroxytryptophan 3,5-Dinitrobenzamide (+)-E2-14:

Triethylamine (83.1 μL, 596 μmol, 2.00 equiv) was added to a solution of12-hydroxytryptophan amine (+)-E2-13 (100 mg, 198 μmol, 1 equiv) and3,5-dinitrobenzoyl chloride (68.7 mg, 298 μmol, 1.50 mmol) indichloromethane (10 mL) at 23° C. After 1 h, saturated aqueous ammoniumchloride solution was added (5 mL) to the reaction mixture. After 5 min,the layers were separated, and the aqueous layer was further extractedwith ethyl acetate (2×5 mL). The combined organic layers were dried overanhydrous sodium sulfate, were filtered, and were concentrated underreduced pressure. The resulting orange foam was purified by flash columnchromatography on silica gel (eluent: gradient, 20-40% ethyl acetate inhexanes) to provide 12-hydroxytryptophan 3,5-dinitrobenzamide (+)-E2-14(129 mg, 93.9%) as a yellow solid. Structural assignments were madeusing additional information from gCOSY, HSQC, and gHMBC experiments. ¹HNMR (600 MHz, CDCl₃, 20° C.): δ 9.20 (s, 1H, Bz-p-H), 8.95 (s, 2H,Bz-o-H), 8.05 (d, J=8.1, 1H, C₈H), 7.98 (d, J=7.6, 1H, C₅H), 7.86 (d,J=7.7, 2H, SO₂Ph-o-H), 7.54 (t, J=7.2, 1H, SO₂Ph-p-H), 7.47 (s, 1H,C₂H), 7.43 (app-t, J=7.5, 2H, SO₂Ph-m-H), 7.39 (app-t, J=7.7, 1H, C₇H),7.35 (app-t, J=7.2, 1H, C₆H), 7.31 (d, J=6.5, 1H, N₁₀H), 5.48 (app-s,1H, C₁₂H), 5.10 (d, J=5.0, 1H, C₁₁H), 4.16 (m, 1H, COCH_(2a)CH₃), 4.06(m, 1H, CO₂CH_(2b)CH₃), 1.14 (app-t, J=7.0, 3H, CO₂CH₂CH₃), 0.93 (s, 9H,Si(CH₃)₂C(CH₃)₃), 0.00 (s, 3H, Si(CH₃)₂C(CH₃)₃), −0.15 (s, 3H,Si(CH₃)₂C(CH₃)₃). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 168.8 (C₁₃), 162.4(C═O_(Bz)), 148.9 (Bz-m-C), 138.2 (SO₂Ph-ipso-C), 137.2 (Bz-ipso-C),135.5 (C₉), 134.1 (SO₂Ph-p-C), 129.4 (SO₂Ph-m-C), 128.6 (C₄), 127.3(Bz-o-C), 126.8 (SO₂Ph-o-C), 125.4 (C₇), 124.2 (C₂), 123.9 (C₆), 122.7(C₃), 121.7 (Bz-p-C), 120.5 (C₅), 114.0 (C₈), 70.1 (C₁₂), 62.3(CO₂CH₂CH₃), 59.3 (C₁₁), 25.7 (Si(CH₃)₂C(CH₃)₃), 18.3 (Si(CH₃)₂C(CH₃)₃),14.2 (CO₂CH₂CH₃), −4.6 (Si(CH₃)₂C(CH₃)₃), −5.1 (Si(CH₃)₂C(CH₃)₃). FTIR(thin film) cm⁻¹: 3394 (br), 2932 (w), 2362 (m), 1736 (m), 1674 (m),1545 (s), 1448 (m), 1345 (s), 1253 (m), 1180 (s), 1119 (m), 1093 (m),977 (w), 920 (w), 838 (m), 726 (m). HRMS (ESI) (m/z): calc'd forC₃₂H₄₀N₅O₁₀SSi [M+NH₄]⁺: 714.2260, found: 714.2251. [α]_(D) ²⁴: +20(c=0.37, CHCl₃). TLC (20% ethyl acetate in hexanes), Rf: 0.21 (UV, CAM).

Dipeptide (+)-E2-S2:

A round-bottom flask was charged sequentially with 12-hydroxytryptophanamine (+)-E2-13 (7.05 g, 14.0 mmol, 1 equiv), N-Boc-sarcosine (3.45 g,18.2 mmol, 1.30 equiv), N-hydroxybenzotriazole (2.84 g, 21.0 mmol, 1.50equiv), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrogen chloride(5.37 g, 28.0 mmol, 2.00 equiv), and powdered 4 Å molecular sieves (4.00g), and the contents were placed under an atmosphere of argon.Dichloromethane (100 mL) was introduced via cannula and the resultingsolution was cooled to 0° C. Triethylamine (5.86 mL, 42.0 mmol, 3.00equiv) was subsequently added dropwise via syringe and the reactionmixture was allowed to warm slowly to 23° C. After 18 h, saturatedaqueous sodium bicarbonate solution (200 mL) was added, and the aqueouslayer was extracted with ethyl acetate (3×250 mL). The combined organiclayers were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The resulting orange foam waspurified by flash column chromatography on silica gel (eluent: 50% ethylacetate in hexanes) to provide dipeptide (+)-E2-S2 (9.05 g, 98%) as ayellow foam. Structural assignments were made using additionalinformation from gCOSY, HSQC, and gHMBC experiments. ¹H NMR (500 MHz,DMSO-d₆, 60° C.): δ 7.97 (d, J=7.7, 1H, N₁₀H), 7.89 (d, J=8.3, 1H, C₈H),7.87 (d, J=7.7, 2H, SO₂Ph-o-H), 7.76 (d, J=7.7, 1H, C₅H), 7.67 (t,J=7.3, 1H, SO₂Ph-p-H), 7.64 (s, 1H, C₂H), 7.55 (app-t, J=7.7, 2H,SO₂Ph-m-H), 7.33 (app-t, J=7.7, 1H, C₇H), 7.26 (app-t, J=7.7, 1H, C₆H),5.16 (d, J=7.7, 1H, C₁₂H), 4.75 (app-t, J=7.8, 1H, C₁₁H), 4.08 (q,J=7.1, 2H, CO₂CH₂CH₃), 3.85-3.65 (m, 1H, C₁₅H_(a)), 3.44 (d, J=17.0, 1H,C₁₅H_(b)), 3.14 (s, 3H, C₁₇H), 1.24 (br-s, 9H, CO₂C(CH₃)₃), 1.17 (t,J=7.1, 3H, CO₂CH₂CH₃), 0.74 (s, 9H, Si(CH₃)₂C(CH₃)₃), −0.04 (s, 3H,Si(CH₃)₂C(CH₃)₃), −0.39 (s, 3H, Si(CH₃)₂C(CH₃)₃). ¹³C NMR (125.8 MHz,DMSO-d₆, 60° C.): δ 171.0 (C₁₃), 169.4 (C₁₆), 156.2 (CO₂C(CH₃)₃), 138.5(SO₂Ph-ipso-C), 136.1 (C₉), 135.6 (SO₂Ph-p-C), 130.9 (SO₂Ph-m-C), 129.6(C₄), 127.6 (SO₂Ph-o-C), 126.1 (C₇), 126.1 (C₂), 124.5 (C₆), 123.6 (C₃),122.1 (C₅), 114.4 (C₈), 80.3 (CO₂C(CH₃)₃), 70.2 (C₁₂), 61.8 (CO₂CH₂CH₃),58.6 (C₁₁), 52.1 (C₁₅), 35.9 (C₁₇), 29.1 (CO₂C(CH₃)₃), 26.5(Si(CH₃)₂C(CH₃)₃), 18.8 (Si(CH₃)₂C(CH₃)₃), 15.0 (CO₂CH₂CH₃), −4.1(Si(CH₃)₂C(CH₃)₃), −4.4 (Si(CH₃)₂C(CH₃)₃). FTIR (thin film) cm⁻¹: 3414(br-m), 2933 (s), 2859 (m), 1739 (m), 1699 (s), 1510 (m), 1450 (m), 1374(s), 1252 (m), 1179 (s), 1120 (m), 1024 (w), 975 (w), 840 (w), 753 (w),685 (w). HRMS (ESI) (m/z): calc'd for C₃₃H₄₈N₃O₈SSi [M+H]⁺: 674.2926,found: 674.2926. [α]_(D) ²⁴: +69 (c=0.24, CHCl₃). TLC (50% ethyl acetatein hexanes), Rf: 0.45 (UV, CAM).

Diketopiperazine (−)-E2-15:

Trifluoroacetic acid (27 mL) was introduced dropwise to a solution ofdipeptide (+)-E2-S2 (14.6 g, 21.7 mmol, 1 equiv) in dichloromethane (140mL) at 23° C. After 1 h, the reaction mixture was concentrated todryness under reduced pressure. The crude residue was dissolved intert-butanol (210 mL). Acetic acid (32 mL) and morpholine (32 mL) weresuccessively added to the solution, and the resulting reaction mixturewas warmed to 80° C. After 1.5 h, the reaction mixture was concentratedunder reduced pressure and the solids were removed by vacuum filtrationover a sintered funnel. The solids were extracted with ethyl acetate andthe combined organic filtrates were concentrated under reduced pressure.The resulting orange oil was purified by flash column chromatography onsilica gel (eluent: gradient, 50→100% ethyl acetate in hexanes) toprovide diketopiperazine (−)-E2-15 (11.1 g, 97.0%) as a yellow foam.Structural assignments were made using additional information fromgCOSY, HSQC, and gHMBC experiments. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ7.97 (d, J=8.4, 1H, C₈H), 7.89 (d, J=7.8, 2H, SO₂Ph-o-H), 7.55 (s, 1H,C₂H), 7.54 (t, J=7.4, 1H, SO₂Ph-p-H), 7.51 (d, J=7.9, 1H, C₅H), 7.47(app-t, J=7.7, 2H, SO₂Ph-m-H), 7.31 (app-t, J=7.6, 1H, C₇H), 7.22(app-t, J=7.8, 1H, C₆H), 6.39 (br-s, 1H, NH), 5.51 (d, J=3.4, 1H, C₁₂H),4.31 (app-s, 1H, C₁₁H), 3.19 (d, J=17.6, 1H, C₁₅H_(a)), 2.32 (s, 3H,C₁₄H), 2.17 (d, J=17.6, 1H, C₁₅H_(b)), 0.89 (s, 9H, Si(CH₃)₂C(CH₃)₃),0.06 (s, 3H, Si(CH₃)₂C(CH₃)₃), −0.07 (s, 3H, Si(CH₃)₂C(CH₃)₃). ¹³C NMR(150 MHz, CDCl₃, 20° C.): δ 164.9 (C₁₃), 162.6 (C₁₆), 138.0(SO₂Ph-ipso-C), 134.7 (C₉), 134.1 (SO₂Ph-p-C), 129.6 (SO₂Ph-m-C), 127.9(C₄), 127.1 (SO₂Ph-o-C), 126.0 (C₂), 125.4 (C₇), 123.6 (C₆), 120.6 (C₃),120.5 (C₅), 113.6 (C₈), 70.4 (C₁₂), 61.7 (C₁₁), 50.5 (C₁₅), 33.3 (C₁₇),25.9 (Si(CH₃)₂C(CH₃)₃), 18.3 (Si(CH₃)₂C(CH₃)₃), −4.7 (Si(CH₃)₂C(CH₃)₃),−5.1 (Si(CH₃)₂C(CH₃)₃). FTIR (thin film) cm⁻¹: 3251 (br m), 2932 (m),1676 (s), 1448 (m), 1371 (m), 1179 (s), 1122 (m), 979 (w), 838 (w), 751(w), 686 (w). HRMS (ESI) (m/z): calc'd for C₂₆H3₄N₃O₅SSi [M+H]⁺:528.1983, found: 528.1982. [α]_(D) ²⁴: −21 (=0.17, CHCl₃). TLC (50%ethyl acetate in hexanes), Rf: 0.32 (UV, CAM).

Tetracyclic Bromide (+)-E2-16:

A solution of bromine (2 M, 61.0 mL, 122 mmol, 4.00 equiv) inacetonitrile that was pre-cooled to 0° C. was poured in one portion intoa solution of diketopiperazine (−)-E2-15 (16.1 g, 30.5 mmol, 1 equiv) inacetonitrile (268 mL) at 0° C. The reaction progress was monitored byTLC analysis in 2 min interval. After 8 min, upon complete consumptionof starting material, anisole (19.9 mL, 183 mmol, 6.00 equiv) that waspre-cooled to 0° C. was poured in one portion into the reaction mixture.After 10 min, a mixture of saturated aqueous sodium thiosulfate solutionand saturated aqueous sodium bicarbonate solution (1:1, 500 mL) wasadded to the red solution. The reaction mixture was extracted with ethylacetate (3×100 mL). The combined organic layers were dried overanhydrous sodium sulfate, were filtered, and were concentrated underreduced pressure. The resulting residue was purified by flash columnchromatography on silica gel (eluent: gradient, 20→50% acetone indichloromethane) to afford a mixture of the endo-tetracyclic bromide(+)-E2-16 and its minor exo-diastereomer (14.2 g, 94.5%, 8.7:1 dr) as awhite foam. The mixture of diastereomers can be separated easily onsmaller scales using the same purification conditions reported here. Ondecagram scales, in some embodiments, it could be more practical tocarry the diastereomeric mixture to next step. Structural assignmentswere made with additional information using gCOSY, HSQC, gHMBC, andNOESY experiments. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ 7.98 (d, J=8.0,2H, SO₂Ph-o-H), 7.54 (d, J=8.3, 1H, C₈H), 7.52 (t, J=7.6, 1H,SO₂Ph-p-H), 7.42 (app-t, J=7.8, 2H, SO₂Ph-m-H), 7.33 (d, J=7.6, 1H,C₅H), 7.28 (app-t, J=7.9, 1H, C₇H), 7.11 (app-t, J=7.6, 1H, C₆H), 6.25(s, 1H, C₂H), 4.62 (d, J=5.9, 1H, C₁₂H), 4.23 (d, J=5.8, 1H, C₁₁H), 4.16(d, J=17.6, 1H, C₁₅H_(a)), 3.91 (br-s, 1H, OH), 3.84 (d, J=17.5, 1H,C₁₅H_(b)), 2.88 (s, 3H, C₁₇H). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ 165.8(C₁₃), 164.9 (C₁₆), 139.2 (C₉), 137.4 (SO₂Ph-ipso-C), 134.0 (SO₂Ph-p-C),131.8 (C₄), 131.2 (C₇), 129.1 (SO₂Ph-m-C), 128.4 (SO₂Ph-o-C), 125.9(C₆), 125.3 (C₅), 116.8 (C₈), 85.1 (C₂), 74.2 (C₁₂), 68.0 (C₃), 63.4(C₁₁), 54.0 (C₁₅), 33.5 (C₁₇). FTIR (thin film) cm⁻¹: 3367 (br s), 2958(s), 1677 (s), 1458 (m), 1399 (m), 1364 (m), 1170 (m), 1089 (w), 729(w), 688 (w). HRMS (ESI) (m/z): calc'd for C₂₀H₁₉BrN₃O₅S [M+H]⁺:492.0223, found: 492.0222. [α]_(D) ²⁴: +56 (c=0.30, CHCl₃). TLC (33%acetone in dichloromethane), Rf: 0.35 (UV, CAM).

Indole-Tethered Tetracycle E2-21:

A solution of n-butyllithium (1.57 M, 18.3 mL, 28.7 mmol, 1.61 equiv) inhexanes was added dropwise to a solution of freshly distilleddiisopropylamine (4.02 mL, 28.7 mmol, 1.61 equiv) in tetrahydrofuran(4.28 mL) at −78° C. After 5 min, the solution was allowed to warm to 0°C. After 35 min, the solution of lithium diisopropylamide wastransferred via cannula to a solution of N-Boc-indole (6.24 g, 28.7mmol, 1.61 equiv) and dimethyldichlorosilane (3.25 mL, 26.8 mmol, 1.50equiv) in tetrahydrofuran (47.3 mL) at 0° C. After 8 h, the reactionmixture was transferred via cannula to a solution of tetracyclic bromide(+)-E2-16 (8.78 g, 17.83 mmol, 1 equiv) and 4-dimethylaminopyridine(4.68 g, 38.28 mmol, 2.15 equiv) in tetrahydrofuran (63.3 mL) at 23° C.After 14 h, the reaction mixture was diluted with ethyl acetate (300 mL)and washed with saturated aqueous sodium bicarbonate solution (500 mL).The aqueous layer was further extracted with ethyl acetate (2×200 mL).The combined organic layers were dried over anhydrous sodium sulfate,were filtered, and were concentrated under reduced pressure. Theresulting orange foam was purified by flash column chromatography onsilica gel (eluent: gradient, 33→50% ethyl acetate in hexanes) toprovide the indole-tethered tetracycle E2-21 (10.1 g, 74.3%) as a whitefoam. Structural assignments were made using additional information fromgCOSY, HSQC, and gHMBC experiments. ¹H NMR (600 MHz, CDCl₃, 20° C.): δ8.03 (d, J=7.6, 2H, SO₂Ph-o-H), 7.96 (d, J=8.3, 1H, C_(8′)H), 7.63 (d,J=7.7, 1H, C_(5′)H), 7.57 (d, J=8.1, 1H, C₈H), 7.53 (t, J=7.4, 1H,SO₂Ph-p-H), 7.44 (app-t, J=7.9, 2H, SO₂Ph-m-H), 7.31 (app-t, J=7.4, 1H,C_(7′)H), 7.27 (app-t, J=7.9, 1H, C₇H), 7.26-7.21 (m, 1H, C₅H),7.26-7.21 (m, 1H, C_(6′)H), 7.26-7.21 (m, 1H, C_(3′)H), 7.00 (app-t,J=7.5, 1H, C₆H), 6.33 (s, 1H, C₂H), 5.13 (d, J=3.7, 1H, C₁₂H), 4.30 (d,J=3.6, 1H, C₁₁H), 4.11 (d, J=17.2, 1H, C₁₅H_(a)), 3.79 (d, J=17.2, 1H,C₁₅H_(b)), 2.82 (s, 3H, C₁₇H), 1.69 (s, 9H, CO₂C(CH₃)₃), 0.54 (s, 3H,Si(CH₃)₂), 0.50 (s, 3H, Si(CH₃)₂). ¹³C NMR (150 MHz, CDCl₃, 20° C.): δ165.5 (C₁₃), 165.2 (C₁₆), 151.7 (C═O_(carbamate)), 139.1 (C_(2′)), 138.9(C₉), 137.8 (SO₂Ph-ipso-C), 137.3 (C_(9′)), 133.8 (SO₂Ph-p-C), 131.4(C_(4′)), 131.3 (C₄), 131.0 (C₇), 129.0 (SO₂Ph-m-C), 128.4 (SO₂Ph-o-C),125.6 (C₆), 125.4 (C₅), 124.9 (C_(7′)), 122.7 (C_(6′)), 121.5 (C_(5′)),121.1 (C_(3′)), 116.5 (C₈), 115.4 (C_(8′)), 85.5 (C₂), 84.6(CO₂C(CH₃)₃), 74.6 (C₁₂), 69.6 (C₃), 66.5 (C₁₁), 54.3 (C₁₅), 33.6 (C₁₇),28.3 (CO₂C(CH₃)₃), 0.5 (Si(CH₃)₂), −0.4 (Si(CH₃)₂). FTIR (thin film)cm⁻¹: 2957 (w), 1715 (s), 1466 (w), 1447 (w), 1373 (s), 1335 (m), 1251(m), 1169 (s), 1072 (w), 922 (w), 834 (w), 751 (m), 689 (w). HRMS (ESI)(m/z): calc'd for C₃₅H₃₈BrN₄O₇SSi [M+H]⁺: 765.1408, found: 765.1441. TLC(50% ethyl acetate in hexanes), Rf: 0.44 (UV, CAM).

Salicylic Tetracycle (+)-E2-22:

A round-bottom flask was charged with indole-tethered tetracycle E2-21(6.85 g, 8.95 mmol, 1 equiv) and 2,6-di-tert-butyl-4-methylpyridine(3.69 g, 17.9 mmol, 2.00 equiv) and azeotropically dried with benzene(3×50 mL) under reduced pressure. The flask was left under reducedpressure for 12 h to ensure the complete removal of benzene thenreturned to atmospheric pressure by backfilling with argon. Anhydrousnitroethane (200 mL, distilled over CaH₂ and stored over 4 Å molecularsieves) was then introduced via cannula and the resulting solution wascooled to 0° C. A solution of silver (I) tetrafluoroborate (8.69 g, 44.8mmol, 5.00 equiv) in nitroethane (30 mL) at 0° C. was introduced viacannula to the solution containing the indole-tethered tetracycle E2-21over 1 min. The reaction mixture immediately changed color to a dark redthen to brown. After 1 h, brine (150 mL) was added and the resultingbiphasic mixture was vigorously stirred at 23° C. After 10 min, thereaction mixture was extracted with ethyl acetate (3×150 mL). Thecombined organic layers were dried over anhydrous sodium sulfate, werefiltered, and were concentrated under reduced pressure. The resultingwhite residue was purified by flash column chromatography (eluent:gradient, 40→100% ethyl acetate in hexanes) to afford the salicylictetracycle (+)-E2-22 (4.20 g, 68.5%) as a white foam. Structuralassignments were made using additional information from gCOSY, HSQC,gHMBC, and NOESY experiments. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 8.25(d, J=8.3, 2H, SO₂Ph-o-H). 7.96 (d, J=8.2, 1H, C₈H), 7.87 (d, J=8.4, 1H, C_(8′)H), 7.74 (t, J=7.5, 1H, SO₂Ph-p-H), 7.59 (app-t, J=7.8, 2H,SO₂Ph-m-H), 7.30 (app-t, J=7.6, 1H, C₇H), 7.15 (app-t, J=7.5, 1H,C_(6′)H), 6.91 (app-t, J=7.5, 1H, C₆H), 6.71 (d, J=7.4, 1H, C₅H), 6.50(app-t, J=7.4, 1H, C_(6′)H), 6.39 (s, 1H, C₂H), 5.51 (d, J=8.0, 1H,C_(5′)H), 4.91 (d, J=9.5, 1H, C₁₂H), 4.32 (d, J=9.5, 1H, C₁₁H), 4.02 (d,J=18.2, 1H, C₁₅H_(a)), 3.95 (d, J=18.1, 1H, C₁₅H_(b)), 3.05 (s, 3H,C₁₇H), 1.70 (s, 9H, CO₂C(CH₃)₃), 0.54 (s, 3H, Si(CH₃)₂), 0.48 (s, 3H,Si(CH₃)₂). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 167.7 (C₁₃), 166.5(C₁₆), 151.6 (C═O_(carbamate)), 139.8 (C₉), 136.8 (C_(9′)), 136.3(SO₂Ph-ipso-C), 136.0 (C_(2′)), 133.8 (SO₂Ph-p-C), 132.0 (C₄), 129.8(C₇), 129.6 (SO₂Ph-m-C), 129.5 (C_(4′)), 128.4 (SO₂Ph-o-C), 125.3(C_(7′)), 125.1 (C_(3′)), 124.5 (C₆), 123.5 (C₅), 122.7 (C_(6′)), 119.3(C_(5′)), 115.5 (C_(8′)), 115.2 (C₈), 85.6 (CO₂C(CH₃)), 80.5 (C₁₂), 79.1(C₂), 61.4 (C₁₁), 56.6 (C₃), 54.2 (C₁₅), 33.5 (C₁₇), 28.3 (CO₂C(CH₃)₃),1.7 (Si(CH₃)₂), 0.6 (Si(CH₃)₂). FTIR (thin film) cm⁻¹: 2927 (s), 1700(m), 1652 (w), 1558 (m), 1494 (m), 1454 (w), 1373 (m), 1328 (w), 1258(w), 1159 (w), 1050 (w), 876 (m), 825 (m), 751 (w). HRMS (ESI) (m/z):calc'd for C₃₅H₃₆N₄NaO₇SSi [M+Na]⁺: 707.1966, found: 707.1986. [α]_(D)²⁴: +203 (c=0.22, CHCl₃). TLC (33% acetone in hexanes), Rf: 0.25 (UV,CAM).

3-Indolylated Tetracycle (+)-E2-24:

Aqueous hydrogen chloride solution (6 N, 20 mL) was added in one portionto a sealed tube charged with a solution of salicylic tetracycle(+)-E2-22 (1.02 g, 1.49 mmol, 1 equiv) in tetrahydrofuran (20 mL) at 23°C. The reaction flask was then sealed and heated to 80° C. After 40 min,the sealed tube was immediately immersed in an ice-water bath andrapidly cooled to 0° C. The cold solution was slowly poured into asaturated aqueous sodium bicarbonate solution (250 mL) at 0° C., andextracted with ethyl acetate (200 mL). The aqueous layer was furtherextracted with ethyl acetate (2×100 mL), and the combined organic layerswere dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The resulting residue was purifiedby flash column chromatography (eluent: gradient, 25→50% acetone indichloromethane) to afford the 3-indolylated tetracycle (+)-E2-24 (456mg, 57.9%) as a white solid. Structural assignments were made usingadditional information from gCOSY, HSQC, gHMBC, and NOESY experiments.¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.94 (br-s, 1H, N_(1′)H), 7.75 (d,J=8.1, 1H, C₈H), 7.44 (dd, J=1.2, 8.6, 2H, SO₂Ph-o-H), 7.37-7.27 (m, 1H,SO₂Ph-p-H), 7.37-7.27 (m, 1H, C_(8′)H), 7.37-7.27 (m, 1H, C₇H),7.18-7.13 (m, 1H, C₅H), 7.18-7.13 (m, 1H, C_(7′)H), 7.09-7.01 (m, 2H,SO₂Ph-m-C), 7.09-7.01 (m, 1H, C₆H), 6.91-6.84 (m, 1H, C_(5′)H),6.91-6.84 (m, 1H, C_(6′)H), 6.64 (d, J=2.7, 1H, C_(2′)H), 6.49 (s, 1H,C₂H), 4.92 (dd, J=3.3, 6.0, 1H, C₁₂H), 4.42 (d, J=5.7, 1H, C₁₁H), 4.09(d, J=17.1, 1H, C₁₅H_(a)), 3.84 (d, J=17.5, 1H, C₁₅H_(b)), 2.88 (s, 3H,C₁₇H), 2.71 (d, J=3.2, 1H, OH). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ166.9 (C₁₃), 165.4 (C₁₆), 139.6 (C₉), 137.4 (SO₂Ph-ipso-C), 136.9(C_(9′)), 134.5 (C₄), 133.0 (SO₂Ph-p-C), 129.6 (C₇), 128.6 (SO₂Ph-m-C),127.5 (SO₂Ph-o-C), 126.2 (C_(4′)), 125.8 (C_(2′)), 125.3 (C₅), 125.2(C₆), 122.8 (C_(7′)), 120.5 (C_(5′)), 120.5 (C_(6′)), 117.3 (C₈), 111.7(C_(8′)), 110.6 (C_(3′)), 82.3 (C₂), 77.4 (C₁₂), 64.6 (C₁₁), 59.8 (C₃),54.3 (C₁₅), 33.5 (C₁₇). FTIR (thin film) cm⁻¹: 3388 (br-s), 3061 (m),2923 (m), 1673 (s), 1458 (m), 1427 (m), 1402 (m), 1357 (m), 1260 (w),1168 (m), 1109 (m), 735 (m), 687 (w). HRMS (ESI) (m/z): calc'd forC₂₈H₂₅N₄O₅S [M+H]⁺: 529.1540, found: 529.1545. [α]_(D) ²⁴: +11 (c=0.05,CHCl₃). TLC (50% acetone in dichloromethane), Rf: 0.41 (UV, CAM).

Bis(Tert-Butoxycarbonyl) Tetracycle (−)-E2-S3:

Di-tert-butyl dicarbonate (783 μL, 3.41 mmol, 4.00 equiv) was added viasyringe to a solution of 3-indolylated tetracycle (+)-E2-24 (450 mg, 852μmol, 1 equiv) and 4-dimethylaminopyridine (52.1 mg, 426 μmol, 0.500equiv) in dichloromethane (10.0 mL) at 23° C. After 30 min, the crudereaction mixture was purified by flash column chromatography on silicagel (eluent: 8% acetone in dichloromethane) to affordbis(tert-butoxycarbonyl) tetracycle (−)-E2-S3 (568 mg, 91.5%) as a whitesolid. Structural assignments were made using additional informationfrom gCOSY, HSQC, and HMBC experiments. ¹H NMR (500 MHz, CDCl₃, 20° C.):δ 7.98 (d, J=7.3, 1H, C_(8′)H), 7.73 (d, J=7.6, 1H, C₈H), 7.67 (d,J=8.1, 1H, C₅H), 7.58 (d, J=7.7, 1H, C_(8′)H), 7.33 (app-t, J=7.9, 1H,C₆H), 7.30 (app-t, J=7.3, 1H, C_(7′)H), 7.25 (app-t, J=8.0, 1H,C_(6′)H), 7.20 (app-t, J=7.5, 1H, C₇H), 7.02 (t, J=7.4, 1H, SO₂Ph-p-H),6.88 (d, J=7.3, 2H, SO₂Ph-o-H), 6.64 (app-t, J=7.5, 2H, SO₂Ph-m-H), 6.58(s, 1H, C₂H), 5.99 (s, 1H, C_(2′)H), 5.92 (d, J=4.0, 1H, C₁₂H), 4.53 (d,J=3.9, 1H, C₁₁H), 4.18 (d, J=17.4, 1H, C₁₅H_(a)), 3.79 (d, J=17.4, 1H,C₁₅H_(b)), 2.81 (s, 3H, C₁₇H), 1.50 (s, 9H, C(CH₃)_(3carbamate)), 0.80(s, 9H, C(CH₃)_(3carbonate)). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ164.8 (C₁₃), 164.5 (C₁₆), 151.4 (C═O_(carbonate)), 148.6(C═O_(carbonate)), 139.2 (C₄), 137.8 (SO₂Ph-ipso-C), 135.8 (C_(9′)),133.8 (C₉), 132.2 (SO₂Ph-p-C), 130.1 (C₆), 128.1 (C_(4′)), 127.7(SO₂Ph-o-C), 127.5 (C_(2′)), 126.8 (C₈), 126.5 (SO₂Ph-o-C), 126.2 (C₇),124.7 (C_(7′)), 122.9 (C_(6′)), 121.5 (C_(5′)), 118.8 (C₅), 115.9(C_(3′)), 115.1 (C_(8′)), 84.0 (C(CH₃)_(3carbamate)), 82.4(C(CH₃)_(3carbonate)), 82.3 (C₂), 78.3 (C₁₂), 63.4 (C₁₁), 59.6 (C₃),54.1 (C₁₅), 33.6 (C₁₇), 28.0 (C(CH₃)_(3carbamate)), 26.6(C(CH₃)_(3carbonate)). FTIR (thin film) cm⁻¹: 2979 (w), 1740 (s), 1708(s), 1686 (s), 1453 (m), 1371 (s), 1280 (s), 1258 (s), 1156 (s), 1100(m), 750 (m). HRMS (ESI) (m/z): calc'd for C₃₈H₄₀N₄NaO₉S [M+Na]⁺:751.2408, found 751.2405. [α]_(D) ²⁴: −22 (c=0.31, CHCl₃). TLC (8%acetone in dichloromethane), Rf: 0.28 (UV, CAM).

Triketopiperazine (−)-E2-25:

Bis(pyridine)silver permanganate (945 mg, 2.45 mmol, 8.00 equiv) wasadded as a solid to a solution of bis(tert-butoxycarbonyl) tetracycle(−)-E2-S3 (224 mg, 307 μmol, 1 equiv) in dichloromethane (10.0 mL) at23° C. After 1 h, the reaction mixture was diluted with dichloromethane(100 mL) and washed with aqueous sodium bisulfite solution (1 M, 125mL). The resulting aqueous layer was extracted with dichloromethane(2×50 mL) and the combined organic layers were dried over anhydroussodium sulfate, were filtered, and were concentrated under reducedpressure. The crude reaction mixture was purified by flash columnchromatography on silica gel (eluent: 50% ethyl acetate in hexanes) toafford triketopiperazine (−)-E2-25 (105 mg, 45.1%) as a white solid.Structural assignments were made using additional information fromgCOSY, HSQC, and HMBC experiments. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ7.97 (d, J.=8.2, 1H, C_(8′)H), 7.86 (d, J=8.0, 1H, C₈H), 7.59-7.52 (m,1H, C₅H), 7.59-7.52 (m, 1H, C₇H), 7.33 (app-t, J=7.6, 1H, C₆H),7.32-7.26 (m, 1H, C_(7′)H), 7.32-7.26 (m, 1H, C_(5′)H), 7.22 (app-t,J=7.1, 1H, C_(6′)H), 7.10 (d, J=7.5, 2H, SO₂Ph-o-H), 6.98 (t, J=7.5, 1H,SO₂Ph-p-H), 6.93 (s, 1H, C₂H), 6.61 (app-t, J=7.9, 2H, SO₂Ph-m-H), 6.53(s, 1H, C_(2′)H), 5.69 (s, 1H, C₁₂H), 4.45 (s, 1H, C₁₁OH), 3.34 (s, 3H,C₁₇H), 1.61 (s, 9H, C(CH₃)_(3carbamate)), 0.78 (s, 9H,C(CH₃)_(3carbonate)). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 165.5 (C₁₅),157.0 (C₁₃), 151.3 (C₁₆), 150.7 (C═O_(carbonate)), 149.0(C═O_(carbamate)), 140.9 (C₉), 136.3 (C_(9′)), 136.2 (SO₂Ph-ipso-C),133.7 (C₄), 132.7 (SO₂Ph-p-C), 130.5 (C₇), 128.0 (SO₂Ph-m-C), 127.4(C_(2′)), 127.4 (C_(4′)), 127.0 (SO₂Ph-o-C), 126.5 (C₆), 125.3 (C₅),124.9 (C_(5′)), 123.3 (C_(6′)), 121.8 (C_(7′)), 120.0 (C₈), 115.7(C_(3′)), 115.2 (C_(8′)), 90.5 (C₁₁), 85.3 (C₁₂), 84.4(C(CH₃)_(3carbonate)), 83.5 (C(CH₃)_(3carbonate)), 82.2 (C₂), 57.8 (C₃),28.3 (C(CH₃)_(3carbamate)), 28.1 (C₁₇), 26.8 (C(CH₃)_(3carbonate)). FTIR(thin film) cm⁻¹: 3386 (br-s), 2981 (w), 1738 (s), 1703 (s), 1453 (m),1371 (s), 1274 (m), 1252 (m), 1156 (m), 751 (m). HRMS (ESI) (m/z):calc'd for C₃₈H₃₉N₄O₁₁S [M+H]⁺: 759.2331, found 759.2302. [α]_(D) ²⁴:−69 (c=0.15, CHCl₃). TLC (40% ethyl acetate in hexanes), Rf: 0.20 (UV,CAM).

Tetracyclic Diol (−)-E2-26:

Sodium borohydride (17.3 mg, 457 μmol, 2.00 equiv) was added as a solidto a solution of triketopiperazine (−)-E2-25 (174 mg, 229 μmol, 1 equiv)in methanol at −20° C. After 20 min, saturated aqueous sodiumbicarbonate solution (5 mL) was added to the reaction mixture. Thesolution was diluted with dichloromethane (60 mL) and washed withsaturated aqueous sodium bicarbonate solution (60 mL). The mixture wasextracted with dichloromethane (2×60 mL), and the combined organiclayers were dried over anhydrous sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The crude reaction mixture waspurified by flash column chromatography on silica gel (eluent: 20%acetone in dichloromethane) to afford tetracyclic diol (−)-E2-26 (130mg, 74.9%) as a white solid. Structural assignments were made usingadditional information from gCOSY, HSQC, and HMBC experiments. ¹H NMR(500 MHz, CDCl₃, 20° C.): δ 7.97 (d, J=7.6, 1H, C_(8′)H), 7.75 (d,J=8.1, 1H, C₈H), 7.52 (d, J=7.1, 1H, C₅H), 7.45 (d, J=7.9, 1H, C_(5′)H),7.36 (app-t, J=7.8, 1H, C₇H), 7.31 (app-t, J=7.3, 1H, C_(7′)H), 7.26(app-t, J=7.6, 1H, C_(6′)H), 7.23 (app-t, J=7.5, 1H, C₆H), 7.01 (d,J=7.4, 2H, SO₂Ph-o-H), 6.97 (t, J=7.5, 1H, SO₂Ph-p-H), 6.94 (s, 1H,C₂H), 6.58 (app-t, J=8.1, 2H, SO₂Ph-m-H), 6.43 (s, 1H, C_(2′)H), 5.66(s, 1H, C₁₂H), 5.39 (d, J=3.0, 1H, C₁₅H), 4.92 (d, J=2.8, 1H, C₁₅OH),4.38 (br-s, 1H, C₁₁OH), 2.99 (s, 3H, C₁₇H), 1.59 (s, 9H,C(CH₃)_(3carbamate)), 0.80 (s, 9H, C(CH₃)_(3carbonate)). ¹³C NMR (125.8MHz, CDCl₃, 20° C.): δ 165.4 (C₁₆), 163.7 (C₁₃), 151.2(C═O_(carbonate)), 149.0 (C═O_(carbamate)), 140.7 (C₉), 136.8(SO₂Ph-ipso-C), 136.3 (C_(9′)), 134.0 (C₄), 132.4 (SO₂Ph-p-C), 129.8(C₇), 127.8 (SO₂Ph-nm-C), 127.7 (C_(4′)), 127.3 (C_(2′)), 126.8(SO₂Ph-o-C), 126.2 (C₆), 125.4 (C₅), 124.8 (C_(7′)), 123.1 (C_(6′)),122.3 (C_(5′)), 119.7 (C₈), 116.0 (C_(3′)), 115.1 (C_(8′)), 90.5 (C₁₁),84.9 (C₁₂), 84.2 (C(CH₃)_(3carbamate)), 83.0 (C(CH₃)_(3carbonate)), 81.4(C₂), 77.1 (C₁₅), 57.3 (C₃), 29.6 (C₁₇), 28.3 (C(CH₃)_(3carbamate)),26.9 (C(CH₃)_(3carbonate)). FTIR (thin film) cm⁻¹:3417 (br-s), 2980 (w),1738 (s), 1687 (s), 1454 (m), 1371 (s), 1276 (m), 1252 (m), 1156 (s),749 (w). HRMS (ESI) (m/z): calc'd for C₃₈H₄₁N₄O₁₁S [M+H]⁺: 761.2487,found 761.2481. [α]_(D) ²⁴: −19 (c=0.16, CHCl₃). TLC (20% acetone indichloromethane), Rf: 0.23 (UV, CAM).

Tetracyclic Dipivaloate (+)-E2-27:

Pivaloyl chloride (75.8 μL, 616 μmol, 4.00 equiv) was added via syringeto a solution of tetracyclic diol (−)-E2-26 (117 mg, 154 μmol, 1 equiv)and 4-dimethylaminopyridine (94.1 mg, 771 μmol, 5.00 equiv) indichloromethane (4 mL) at 23° C. After 1 h 45 min, methanol (50 μL) wasadded to the reaction mixture, and the crude solution was purified byflash column chromatography on silica gel (eluent: 1% acetone indichloromethane) to afford tetracyclic dipivaloate (+)-E2-27 (119 mg,83.0%) as a white solid. Structural assignments were made usingadditional information from gCOSY, HSQC, and HMBC experiments. ¹H NMR(500 MHz, CDCl₃, 20° C.): δ 7.96 (d. J=7.9, 1H, C_(8′)H), 7.81 (d,J=8.1, 1H, C₈H), 7.60 (dd, J=0.7, 7.5, 1H, C₅H), 7.45 (br-s, 1H,C_(5′)H), 7.39 (app-dt, J=1.2, 7.7, 1H, C₇H), 7.32-7.25 (m, 1H, C₆H),7.32-7.25 (m, 1H, C_(7′)H), 7.20-7.13 (m, 2H, SO₂Ph-o-H), 7.20-7.13 (m,1H, C_(6′)H), 6.99 (t, J=7.5, SO₂Ph-p-H), 6.91 (s, 1H, C₂H), 6.63(app-t, J=7.9, 2H, SO₂Ph-m-H), 6.52 (br-s, 1H, C₁₅H), 6.44 (s, 1H,C_(2′)H), 5.70 (s, 1H, C₁₂H), 2.91 (s, 3H, C₁₇H), 1.59 (s, 9H,C(CH₃)_(3carbamate)), 1.40 (s, 9H, C(CH₃)_(3pivaloate)), 0.79 (s, 9H,C(CH₃)_(3carbonate)), 0.68 (s, 9H, C(CH₃)_(3pivaloate)). ¹³C NMR (125.8MHz, CDCl₃, 20° C.): δ 177.3 (C═O_(pivaloate)), 176.3 (C═O_(pivaloate)),161.3 (C₁₆), 160.8 (C₁₃), 151.0 (C═O_(carbonate)), 149.0(C═O_(carbamate)), 141.5 (C₉), 136.6 (SO₂Ph-ipso-C), 136.3 (C_(9′)),133.1 (C₄), 132.6 (SO₂Ph-p-C), 130.0 (C₇), 127.9 (SO₂Ph-m-C), 127.5(C_(4′)), 127.3 (C_(2′)), 127.0 (SO₂Ph-o-C), 126.0 (C₆), 125.7 (C₅),124.7 (C_(7′)), 123.0 (C_(6′)), 122.3 (C_(5′)), 119.4 (C₈), 116.6(C_(3′)), 115.0 (C_(8′)), 92.2 (C₁₁), 85.2 (C₁₂), 84.2(C(CH₃)_(3carbonate)), 83.2 (C₂), 82.8 (C(CH₃)_(3carbonate)), 78.3(C₁₅), 57.9 (C₃), 39.3 (C(CH₃)_(3pivaloate)), 38.7(C(CH₃)_(3pivaloate)), 30.7 (C₁₇), 28.3 (C(CH₃)_(3carbamate)), 27.2(C(CH₃)_(3pivaloate)), 26.8 (C(CH₃)_(3carbonate)), 26.3(C(CH₃)_(3pivaloate)). FTIR (thin film) cm⁻¹: 2978 (m), 1742 (s), 1699(m), 1454 (m), 1371 (s), 1276 (m), 1252 (m), 1155 (m), 1123 (s), 750(m). HRMS (ESI) (m/z): calc'd for C₄₈H₅₆N₄NaO₁₃S [M+Na]⁺: 951.3457,found 951.3451. [α]_(D) ²⁴: +5.3 (c=0.095, CHCl₃). TLC (1% acetone indichloromethane), Rf: 0.27 (UV, CAM).

Tetracyclic Bisthioether (+)-E2-37:

Trifluoroacetic acid (3 mL) was added via syringe to a solution oftetracyclic dipivaloate (+)-E2-27 (109 mg, 117 μmol, 1 equiv) and3-mercapto-2-butanone (366 mg, 3.51 mmol, 30.0 equiv, Ross, N. C.;Levine, R. J. Org. Chem. 1964, 29, 2346) in nitromethane (3 mL) at 23°C. After 45 min, the reaction mixture was diluted with dichloromethane(60 mL) and washed with saturated aqueous sodium bicarbonate solution(100 mL). The aqueous layer was extracted with dichloromethane (2×30mL), and the combined organic layers were dried over anhydrous sodiumsulfate, were filtered, and were concentrated under reduced pressure.The crude reaction mixture was purified by flash column chromatographyon silica gel (eluent: 20% acetone in dichloromethane) to afford adiastereomeric mixture of bisthioethers (68.8 mg, 80.2%, 3:1 dr,major:minor) as a beige solid. A 25-mL Pyrex pear-shaped flask wassequentially charged with the diastereomeric mixture of bisthioethers(68.8 mg, 93.9 μmol, 1 equiv), L-ascorbic acid (165 mg, 939 μmol, 10.0equiv), sodium L-ascorbate (186 mg, 939 μmol, 10.0 equiv), and1,4-dimethoxynaphthalene (1.06 g, 5.63 mmol, 60.0 equiv), and themixture was placed under an argon atmosphere. Acetonitrile (8 mL) andwater (2 mL) were then sequentially introduced via syringe and theresulting solution was sparged with argon for 5 min at 23° C. The systemwas stirred vigorously and irradiated in a Rayonet photoreactor equippedwith 16 radially distributed (r=12.7 cm) 25 W black light phosphor lamps(λ_(max)=350 nm) at 25° C. After 1.5 h, the lamps were turned off andthe reaction mixture was diluted with dichloromethane (60 mL). Theresulting solution was washed with saturated aqueous sodium bicarbonatesolution (60 mL). The aqueous layer was extracted with dichloromethane(2×10 mL), and the combined organic layers were dried over anhydroussodium sulfate, were filtered, and were concentrated under reducedpressure. The crude reaction mixture was purified by flash columnchromatography on silica gel (eluent: gradient, 20->25% acetone indichloromethane) to afford tetracyclic bisthioether (+)-E2-37 (31.0 mg,55.7%) as a single diastereomer and as a clear film. Structuralassignments were made using additional information from gCOSY, HSQC, andHMBC experiments. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 8.00 (s, 1H,N_(1′)H), 7.85 (d, J=7.4, 1H, C_(5′)H), 7.37 (d, J=7.3, 1H, C₅H), 7.31(d, J=7.5, 1H, C_(8′)H), 7.17 (app-t, J=7.2, 1H, C_(7′)H), 7.14 (app-t,J=7.2, 1H, C_(6′)H), 7.09 (d, J=2.5, 1H, C_(2′)H), 7.05 (app-t, J=7.5,1H, C₇H), 6.69 (app-t, J=7.3, 1H, C₆H), 6.62 (d, J=7.7, 1H, C₈H), 6.29(s, 1H, C₂H), 5.30 (d, J=1.8, 1H, C₁₂H), 5.07 (s, 1H, N₁H), 4.76 (s, 1H,C₁₅H), 3.34 (d, J=2.0, 1H, C₁₂OH), 3.10-3.02 (m, 2H, C₂₂H), 3.08 (s, 3H,C₁₇H), 2.98-2.86 (m, 2H, C₂₃H), 2.86-2.77 (m, 1H, C₁₈H_(a)), 2.75-2.67(m, 1H, C₁₈H_(b)), 2.47-2.32 (m, 2H, C₁₉H), 2.20 (s, 3H, C₂₅H), 2.04 (s,3H, C₂₁H). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ 206.9 (C₂₄), 206.3(C₂₀), 165.4 (C₁₆), 165.1 (C₁₃), 147.5 (C₉), 137.1 (C_(9′)), 132.2 (C₄),128.8 (C₇), 125.8 (C_(4′)), 123.7 (C₅), 123.2 (C_(2′)), 122.4 (C_(7′)),121.2 (C_(5′)), 120.0 (C_(6′)), 119.3 (C₆), 114.9 (C_(3′)), 111.8(C_(8′)), 110.2 (C₈), 81.6 (C₂), 81.5 (C₁₂), 73.5 (C₁₁), 66.9 (C₁₅),59.3 (C₃), 44.1 (C₂₃), 42.6 (C₁₉), 32.0 (C₁₇), 30.3 (C₂₅), 30.1 (C₂₁),28.6 (C₂₂), 25.7 (C₁₈). FTIR (thin film) cm⁻¹: 3371 (br-s), 2930 (w),1711 (m), 1661 (s), 1423 (m), 1397 (m), 1364 (w), 1238 (w), 743 (m).HRMS (ESI) (m/z): calc'd for C₃₀H₃₂N₄NaO₅S₂ [M+Na]⁺: 615.1706, found615.1693. [α]_(D) ²⁴: +179 (c=0.075, CHCl₃). TLC (25% acetone indichloromethane), Rf: 0.23 (UV, CAM).

(+)-Bionectin A (E2-1):

Pyrrolidine (20 μL, 242 μmol, 27.6 equiv) was added via syringe to asolution of tetracyclic bisthioether (+)-E2-37 (5.2 mg, 8.77 μmol, 1equiv) and ethanethiol (20 μL, 270 μmol, 30.8 equiv) in tetrahydrofuran(1 mL) at 23° C. After 2 h, the reaction mixture was diluted withdichloromethane (10 mL) and washed with aqueous hydrochloric acid (1N,10 mL). The aqueous layer was extracted with dichloromethane (2×5 mL),and the combined organic layers were dried over anhydrous sodiumsulfate, were filtered, and were concentrated under reduced pressure.The crude reaction mixture was purified by flash column chromatographyon silica gel (eluent: 10% acetone in dichloromethane) to afford(+)-bionectin A (E2-1) (3.2 mg, 81%) as a beige solid. Structuralassignments were made using additional information from gCOSY, HSQC,HMBC, and NOESY experiments. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 8.11(br-s, 1H, N_(1′)H), 7.91 (d, J=7.8, 1H, C_(5′)H), 7.44 (d, J=7.5, 1H,C₅H), 7.32 (d, J=8.1, 1H, C_(8′)H), 7.18 (app-t, J=6.9, 1H, C_(7′)H),7.16-7.11 (m, 1H, C_(6′)H), 7.16-7.11 (m, 1H, C₇H), 7.06 (d, J=2.6, 1H,C_(2′)H), 6.83 (app-t, J=7.5, 1H, C₆H), 6.69 (d, J=7.8, 1H, C₈H), 6.30(s, 1H, C₂H), 5.36 (br-s, 1H, N₁H), 5.32 (s, 1H, C₁₂H), 5.19 (s, 1H,C₁₂OH), 5.17 (s, 1H, C₁₅H), 3.11 (s, 3H, C₁₇H). ¹³C NMR (125.8 MHz,CDCl₃, 20° C.): δ 166.2 (C₁₃), 161.9 (C₁₆), 147.3 (C₉), 137.1 (C_(9′)),130.9 (C₄), 129.6 (C₇), 126.3 (C_(4′)), 124.7 (C₅), 123.5 (C_(2′)),122.6 (C_(7′)), 121.6 (C_(5′)), 120.1 (C_(6′)), 120.1 (C₆), 113.4(C_(3′)), 111.7 (C_(8′)), 110.9 (C₈), 82.5 (C₂), 81.0 (C₁₂), 77.0 (C₁₁),67.7 (C₁₅), 61.6 (C₃), 32.0 (C₁₇). FTIR (thin film) cm⁻¹: 3402 (br-s),2922 (w), 1677 (s), 1482 (w), 1458 (w), 1377 (m), 1237 (m), 1093 (w),747 (m). HRMS (ESI) (m/z): calc'd for C₂₂H₁₉N₄O₃S₂ [M+H]⁺: 451.0893,found 451.0891. [α]_(D) ²⁴: +284 (c=0.11, CHCl₃). TLC (10% acetone indichloromethane), Rf: 0.25 (UV, CAM).

(+)-Bionectin C (E2-2):

Sodium borohydride (2.2 mg, 57.7 μmol, 10.0 equiv) was added as a solidto a solution of (+)-bionectin A (E2-1) (2.6 mg, 5.77 μmol, 1 equiv) andmethyl iodide (35.9 μL, 577 μmol, 100 equiv) in pyridine (100 μL) andmethanol (200 μL) at 0° C. After 1 h, the reaction mixture was dilutedwith ethyl acetate (10 mL) and washed with saturated aqueous ammoniumchloride solution (10 mL). The aqueous layer was extracted with ethylacetate (2×10 mL), and the combined organic layers were dried overanhydrous sodium sulfate, were filtered, and were concentrated underreduced pressure. The crude reaction mixture was purified by flashcolumn chromatography on silica gel (eluent: gradient, 10→15% acetone indichloromethane) to afford (+)-bionectin C (E2-2) (2.7 mg, 97%) as awhite solid. Structural assignments were made using additionalinformation from gCOSY, HSQC, HMBC, and NOESY experiments. ¹H NMR (500MHz, CDCl₃, 20° C.): δ 8.00 (br-s, 1H, N_(1′)H), 7.85 (d, J=7.9, 1H,C_(5′)H), 7.41 (d, J=7.5, 1H, C₅H), 7.31 (d, J=7.6, 1H, C_(8′)H), 7.17(app-dt, J=1.0, 7.1, 1H, C_(7′)H), 7.13 (app-dt, J=1.0, 7.1, 1H,C_(6′)H), 7.10 (d, J=2.7, 1H, C_(2′)H), 7.08 (app-dt, J=1.1, 7.6, 1H,C₇H), 6.71 (ap-dt, J=0.7, 7.5, 1H, C₆H), 6.63 (d, J=7.8, 1H, C₈H), 6.34(s, 1H, C₂H), 5.30 (d, J=2.1, 1H, C₁₂H), 5.09 (s, 1H, N₁H), 4.59 (s, 1H,C₁₅H), 3.24 (d, J=2.2, 1H, C₁₂OH), 3.10 (s, 3H, C₁₇H), 2.45 (s, 3H,C₁₅SCH₃), 2.06 (s, 3H, C₁₁SCH₃). ¹³C NMR (125.8 MHz, CDCl₃, 20° C.): δ165.0 (C₁₆), 164.8 (C₁₃), 147.6 (C₉), 137.1 (C_(9′)), 131.9 (C₄), 129.0(C₇), 126.0 (C_(4′)), 123.4 (C₅), 123.1 (C_(2′)), 122.6 (C_(7′)), 121.3(C_(5′)), 120.1 (C_(6′)), 119.3 (C₆), 115.1 (C_(3′)), 111.8 (C_(8′)),110.1 (C₈), 81.8 (C₂), 80.5 (C₁₂), 73.4 (C₁₁), 67.8 (C₁₅), 59.1 (C₃),32.4 (C₁₇), 18.3 (C₁₅SCH₃), 15.7 (C₁₁SCH₃). FTIR (thin film) cm⁻¹: 3399(br-s), 2921 (w), 1661 (s), 1608 (w), 1483 (w), 1458 (w), 1424 (m), 1397(m), 1238 (m), 1087 (w), 741 (m). HRMS (ESI) (m/z): calc'd forC₂₄H₂₅N₄O₃S₂ [M+H]⁺: 481.1363, found 481.1355. [α]_(D) ²⁴: +270(c=0.009, CHCl₃). TLC (10% acetone in dichloromethane), Rf: 0.08 (UV,CAM).

Bionectin A p-Nitrobenzoate (E2-38):

p-Nitrobenzoyl chloride (1.6 mg, 8.67 μmol, 1.50 equiv) was added to asolution of (+)-bionectin A (E2-1) (2.6 mg, 5.78 μmol, 1 equiv) and4-dimethylaminopyridine (3.5 mg, 28.9 μmol, 5.00 equiv) indichloromethane (1 mL) at 0° C. After 1 h, another portion ofp-nitrobenzoyl chloride (1.5 mg, 8.08 μmol, 1.40 equiv) was added to thereaction. After 1 h 10 min, another portion of p-nitrobenzoyl chloride(2.2 mg, 11.9 μmol, 2.05 equiv) was added to the reaction. After 1 h 20min, another portion of 4-dimethylaminopyridine (0.7 mg, 5.73 μmol, 0.99equiv) was added to the reaction. After 1 h 10 min, p-nitrobenzoylchloride (5.0 mg, 26.9 μmol, 4.66 equiv) was added to the reaction.After 38 min, methanol (50 μL) was added to the reaction. The crudereaction mixture was purified by flash column chromatography on silicagel (eluent: 2% acetone in dichloromethane) to afford (+)-bionectin Ap-nitrobenzoate (E2-38) (3.4 mg, 98%) as a yellow solid. Crystalssuitable for X-ray diffraction were obtained by slow evaporation of asaturated solution in chloroform at 23° C. ¹H NMR (500 MHz, CDCl₃, 20°C.): δ 8.05 (br-s, 1H, N_(1′)H), 8.04 (d, J=8.8, 2H, p-NO₂Bz-m-H), 7.75(d, J=8.2, 1H, C_(5′)H), 7.72 (d, J=8.9, 2H, p-NO₂Bz-o-H), 7.66 (d,J=7.4, C₅H), 7.21 (app-t, J=7.7, 1H, C₇H), 7.19 (d, J=8.1, 1H, C_(8′)H),7.04 (d, J=2.7, 1H, C_(2′)H), 7.00 (app-t, J=7.9, 1H, C_(7′)H), 6.95(app-t, J=7.6, 1H, C₆H), 6.91 (app-t, J=7.3, 1H, C_(6′)H), 6.73 (d,J=7.8, 1H, C₈H), 6.65 (s, 1H, C₁₂H), 6.55 (s, 1H, C₂H), 5.40 (br-s, 1H,N₁H), 5.26 (s, 1H, C₁₅H), 3.03 (s, 3H, C₁₇H). ¹³C NMR (125.8 MHz, CDCl₃,20° C.): δ 163.7 (C═O_(Bz)), 163.1 (C₁₃), 161.7 (C₁₆), 150.4(p-NO₂Bz-p-C), 147.7 (C₉), 137.1 (C_(9′)), 134.9 (p-NO₂Bz-ipso-C), 130.9(p-NO₂Bz-m-C), 130.1 (C₇), 129.5 (C₄), 125.7 (C_(4′)), 125.0 (C₅), 124.7(C_(2′)), 123.3 (p-NO₂Bz-o-C), 122.7 (C_(7′)), 121.1 (C_(5′)), 120.2(C₆), 120.1 (C_(6′)), 111.8 (C_(8′)), 111.8 (C_(3′)), 110.9 (C₈), 81.3(C₂), 80.6 (C₁₂), 75.8 (C₁₁), 68.7 (C₁₅), 60.9 (C₃), 32.3 (C₁₇). FTIR(thin film) cm⁻¹: 3406 (br-s), 1738 (w), 1691 (s), 1608 (w), 1526 (m),1346 (w), 1263 (m), 1099 (m), 749 (m), 715 (w). HRMS (ESI) (m/z): calc'dfor C₂₉H₂₁N₅NaO₆S₂ [M+Na]⁺: 622.0825, found 622.0820. [α]_(D) ²⁴: +17(c=0.12, CHCl₃). TLC (2% acetone in dichloromethane), Rf: 0.28 (UV,CAM).

3-Mercaptopropiophenone (E2-30):

Triethylamine (1.49 mL, 10.7 mmol, 1.50 equiv) was added to a solutionof 3-chloropropiophenone (1.20 g, 7.12 mmol, 1 equiv) in dichloromethane(100 mL) at 23° C. Thioacetic acid (602 μL, 8.54 mmol, 1.20 equiv) wasthen added dropwise to the solution. After 1 h, the reaction mixture wasconcentrated in vacuo. The crude residue was dissolved intetrahydrofuran (50 mL) and aqueous hydrochloric acid (6 N, 50 mL) wasadded to the solution. The reaction mixture was then heated to reflux.After 36 h, the reaction was diluted with ethyl acetate (200 mL) andwashed with saturated aqueous sodium bicarbonate solution (400 mL). Theaqueous layer was extracted with ethyl acetate (3×200 mL), and thecombined organic layers were dried over sodium sulfate, were filtered,and were concentrated under reduced pressure. The crude reaction mixturewas purified by flash column chromatography on silica gel (eluent: 20%ethyl acetate in hexanes) to afford 3-mercaptopropiophenone (E2-30, 703mg, 59.4%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.93(d, J=7.5, 2H, COPh-o-H), 7.55 (t, J=7.5, 1H, COPh-p-H), 7.45 (app-t,J=7.5, 2H, COPh-m-H), 3.31 (t, J=7, 2H, CH₂CH₂SH, 2.89 (dt, J=8.5, 6.0,2H, CH₂CH₂SH), 1.74 (t, 0.1=8.5, 1H, SH). ¹³C NMR (125.8 MHz, CDCl₃, 20°C.): δ 198.2 (C═O), 136.8 (COPh-ipso-C), 133.6 (COPh-p-C), 128.9(COPh-m-C), 128.2 (COPh-o-C), 42.7 (CH₂CH₂SH), 19.1 (CH₂CH₂SH). FTIR(thin film) cm⁻¹: 3061 (w), 2941 (w), 1683 (s), 1597 (m), 1580 (m) 1448(m). HRMS (ESI) (m/z): calc'd for C₉H₁₁OS [M+H]⁺: 167.0525, found167.0526. TLC (20% ethyl acetate in hexanes), Rf: 0.28 (UV, CAM).

Bisproline Bis(Ethylmethylketone Thioether) (−)-E2-31:

Trifluoroacetic acid (15 mL) was added via syringe to a solution ofbisproline diol E2-S4 (397 mg, 1.76 mmol, 1 equiv) and3-mercaptobutan-2-one (E2-29, 928 μL, 8.77 mmol, 5.00 equiv) inacetonitrile (15 mL) at 23° C. The clear solution immediately turnedyellow. After 30 min, the reaction was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium bicarbonate solution (50mL). The aqueous layer was extracted with ethyl acetate (3×20 mL), andthe combined organic layers were dried over sodium sulfate, werefiltered, and were concentrated under reduced pressure. The crudereaction mixture was purified by flash column chromatography on silicagel (eluent: 3% acetone in dichloromethane) to afford the bisprolinebis(ethylmethylketone thioether) (−)-E2-31 (490 mg, 70.2%) as a whitesolid. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 3.68-3.62 (m, 2H, C₂H),3.56-3.51 (m, 2H, C₂H), 2.90-2.87 (m, 4H, C₈H), 2.75-2.61 (m, 4H, C₇H),2.46-2.42 (m, 2H, C₄H_(a)), 2.33-2.22 (m, 2H, C₃H_(a)), 2.12-2.04 (m,2H, C₄H_(b)), 2.10 (s, 6H, COCH₃), 2.01-1.95 (m, 2H, C₃H_(b)). ¹³C NMR(125.8 MHz, CDCl₃, 20° C.): δ 206.4 (C₉), 165.3 (C₆), 71.7 (C₅), 45.4(C₂), 43.2 (C₇), 35.4 (C₄), 30.0 (COCH₃), 25.2 (C₈), 20.0 (C₃). FTIR(thin film) cm⁻¹: 1715 (m), 1660 (s), 1409 (s), 1363 (w), 1158 (w). HRMS(ESI) (m/z): calc'd for C₁₈H₃₀N₃O₄S₂ [M+NH₄]⁺: 416.1672, found:416.1679. [α]_(D) ²⁴: −33 (c=0.28, CH₂Cl₂). TLC (10% acetone indichloromethane), Rf: 0.39 (UV, CAM).

Bisproline Epidithiodiketopiperazine (−)-E2-34:

Pyrrolidine (70.0 μL, 852 μmol, 4.07 equiv) was added to a solution ofbis(ethylmethylketone thioether) (−)-E2-31 (83.5 mg, 210 μmol, 1 equiv)in acetonitrile (250 μL) at 23° C., and the reaction was placed under aballoon of oxygen. The clear solution immediately turned orange. After 1h, the reaction was diluted with dichloromethane (5 mL) and washed withsaturated aqueous ammonium chloride solution (5 mL). The aqueous layerwas extracted with ethyl acetate (3×3 mL), and the combined organiclayers were dried over sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The orange residue was purified byflash column chromatography on silica gel (eluent: 3% acetone indichloromethane) to afford the bisproline epidithiodiketopiperazine(−)-E2-34 (34.8 mg, 64.8%) as a white solid. ¹H NMR (500 MHz, CDCl₃, 20°C.): δ 3.88-3.84 (m, 2H, C₂H_(a)), 3.58-3.52 (m, 2H, C₁₂H_(b)),3.02-2.94 (m, 2H, C₄H_(a)), 2.35-2.27 (m, 2H, C₄H_(b)), 2.35-2.27 (m,2H, C₃H_(a)), 2.25-2.18 (m, 2H, C₃H_(b)). ¹³C NMR (125.8 MHz, CDCl₃, 20°C.): δ 164.1 (C₆), δ 78.1 (C₅), δ 46.6 (C₂), δ 32.9 (C₄), δ 24.4 (C₃).FTIR (thin film) cm⁻¹: 2921 (m), 1660 (s), 1405 (m), 1338 (w), 1097 (m).HRMS (ESI) (m/z): calc'd for C₁₀H₁₂N₂NaO₂S₂ [M+Na]⁺: 279.0323, found:279.0314. [α]_(D) ²⁴: −144 (c=0.11, CH₂Cl₂). TLC (10% acetone indichloromethane), Rf: 0.44 (UV, CAM).

Bisproline bis(Ethylphenylketone Thioether) (−)-E2-32:

Trifluoroacetic acid (1 mL) was added via syringe to a solution ofbisproline diol E2-S4 (36.6 mg, 0.162 mmol, 1 equiv) and3-mercaptopropiophenone (E2-30, 76.2 μL, 801 μmol, 5.00 equiv) inacetonitrile (1 mL) at 23° C. The clear solution immediately turnedyellow. After 30 min, the reaction was diluted with ethyl acetate (5 mL)and washed with saturated aqueous sodium bicarbonate solution (5 mL).The aqueous layer was extracted with ethyl acetate (3×5 mL), and thecombined organic layers were dried over sodium sulfate, were filtered,and were concentrated under reduced pressure. The crude reaction mixturewas purified by flash column chromatography on silica gel (eluent: 3%acetone in dichloromethane) to afford the bisprolinebis(ethylmethylketone thioether) (−)-E2-32 (65.9 mg, 77.5%) as a whitesolid. ¹H NMR (500 MHz, CDCl₃, 20° C.):δ 7.89 (d, J=7.5, 4H, COPh-o-H),7.53 (t, J=7.5, 2H, COPh-p-H), 7.42 (app-t, J=8.0, 4H, COPh-m-H),3.73-3.67 (m, 2H, C₂H_(a)), 3.61-3.56 (m, 2H, C₁₂H_(b)), 3.33-3.27 (m,2H, C₇H_(a)), 3.23-3.26 (m, 2H, C₇H_(b)), 3.12-3.09 (m, 4H, C₈H),2.55-2.51 (m, 2H, C₄H_(a)), 2.38-2.28 (m, 2H, C₃H_(a)), 2.17-2.10 (m,2H, C₄H_(b)), 2.05-1.99 (m, 2H, C₃H_(b)). ¹³C NMR (125.8 MHz, CDCl₃, 20°C.): δ 198.7 (C₉), 166.2 (C₆), 137.2 (COPh-ipso-C), 133.6 (COPh-p-C),128.9 (COPh-m-C), 128.2 (COPh-o-C), 72.5 (C₅), 45.7 (C₂), 38.9 (C₇),35.7 (C₄), 25.7 (C₈), 20.1 (C₃). FTIR (thin film) cm⁻¹: 2956 (w), 1683(m), 1660 (m), 1597 (w), 1448 (w), 1406 (m), 1350 (w). HRMS (ESI) (m/z):calc'd for C₂₈H₃₄N₃O₄S₂ [M+NH₄]⁺: 540.1925, found: 540.1925. [α]_(D) ²⁴:−54 (c=0.17, CH₂Cl₂). TLC (10% acetone in dichloromethane), Rf: 0.43(UV, CAM).

Bisproline Epidithiodiketopiperazine (−)-E2-34:

Pyrrolidine (24.3 μL, 284 μmol, 3.79 equiv) was added to a solution ofbis(ethylmethylketone thioether) (−)-E2-32 (39.2 mg, 75.0 μmol, 1 equiv)in acetonitrile (250 μL) at 23° C., and the reaction was placed under aballoon of oxygen. The clear solution immediately turned orange. After 1h, the reaction was diluted with dichloromethane (5 mL) and washed withsaturated aqueous ammonium chloride solution (5 mL). The aqueous layerwas extracted with dichloromethane (3×3 mL), and the combined organiclayers were dried over sodium sulfate, were filtered, and wereconcentrated under reduced pressure. The orange residue was purified byflash column chromatography on silica gel (eluent: 3% acetone indichloromethane) to afford the bisproline epidithiodiketopiperazine(−)-E2-34 (11.5 mg, 59.8%) as a white solid.

3-Propyl Tetracyclic Bis(Ethylphenylketone Thioether) (+)-E2-33:

Trifluoroacetic acid (1 mL) was added via syringe to a solution of3-propyl tetracyclic diol E2-S5 (46.3 mg, 95.4 μmol, 1 equiv) and3-mercaptopropiophenone (E2-30, 72.3 μL, 477 μmol, 5.00 equiv) inacetonitrile (1 mL) at 23° C. The clear solution immediately turnedyellow. After 30 min, the reaction was diluted with ethyl acetate (5 mL)and washed with saturated aqueous sodium bicarbonate solution (2 mL).The aqueous layer was extracted with ethyl acetate (3×5 mL), and thecombined organic layers were dried over sodium sulfate, were filtered,and were concentrated under reduced pressure. The crude reaction mixturewas purified by flash column chromatography on silica gel (eluent: 3%acetone in dichloromethane) to afford the 3-propyl tetracyclicbis(ethylphenylketone thioether) (+)-E2-33 (54.5 mg, 73.1%) as a whitesolid. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 8.00-7.97 (m, 4H, COPh-m-H),7.81 (d, J=8.5, 2H, SO₂Ph-o-H), 7.67 (d, 1H, C₈H), (7.53-7.48, (m, 1H,SO₂Ph-p-H), 7.53-7.48 (m, 2H, SO₂Ph-m-H), 7.47-7.53 (m, 4H, COPh-o-H),7.47-7.35 (m, 2H, COPh-p-H), 7.53-7.48 (m, 1H, SO₂Ph-p-H), 7.17 (app-dt,J=1.3, 7.0, 1H, C₇H), 7.06 (d, J=7.5, 1H, C₅H), 7.00 (app-t, J=7.5, 1H,C₆H), 6.26 (s, 1H, C₂H), 3.45-3.38 (m, 1H, C₁₉H_(a)), 3.30-3.23 (m, 1H,C₁₉H_(b)), 3.12-3.02 (m, 2H, C₂₀H), 3.07 (s, 3H, C₁₈H), 2.99-2.92 (m,2H, C₂₂H), 2.81 (d, J=14, 1H, C₁₂H_(a)), 2.61-2.68 (m, 2H, C₂₃H), 2.30(d, J=14, 1H, C₁₂H_(b)), 1.92 (s, 3H, C₁₇H), 1.44-1.37 (m, 2H,CH₂CH₂CH₃), 1.31-1.22 (m, 2H, CH₂CH₂CH₃), 0.56-0.52 (m, 3H, CH₂CH₂CH₃).¹³C NMR (125 MHz, CDCl₃, 20° C.): δ 199.1 (C₂₁), 198.4 (C₂₄), 166.7(C₁₆), 164.8 (C₁₃), 143.6 (C₉), 138.9 (SO₂Ph-ipso-C), 137.2(COPh-ipso-C), 137.1 (COPh-ipso-C), 136.3 (C₄), 133.9 (SO₂Ph-p-C), 133.9(COPh-p-C), 133.8 (COPh-p-C), 129.9 (C₇), 129.4 (SO₂Ph-o-C), 129.3(SO₂Ph-m-C), 129.2 (COPh-o-C), 129.0 (COPh-o-C), 128.8 (COPh-m-C), 128.1(COPh-m-C), 125.3 (C₆), 123.5 (C₅), 116.6 (C₈), 83.0 (C₂), 71.7 (C₁₁),68.8 (C₁₅), 54.1 (C₃), 50.0 (C₁₂), 42.5 (CH₂CH₂CH₃), 39.6 (C₂₀), 38.8(C₂₃), 30.1 (C₁₈), 27.1 (C₁₇), 25.9 (C₁₉), 25.3 (C₂₂), 38.4 (CH₂CH₂CH₃),14.7 (CH₂CH₂CH₃). FTIR (thin film) cm⁻¹: 2924 (m), 2851 (m), 1682 (s),1597 (w), 1448 (m), 1372 (m). HRMS (ESI) (m/z): calc'd for C₄₂H₄₇N₄O₆S₃[M+NH₄]⁺: 799.2652, found: 799.2658. [α]_(D) ²⁴: +124 (c=0.075). TLC (5%acetone in dichloromethane), Rf: 0.25 (UV, CAM).

3-Propyl Pentacyclic Epidithiodiketopiperazine E2-35:

Pyrrolidine (6.8 μL, 82.8 μmol, 4.16 equiv) was added to a solution of3-propyl tetracyclic bis(ethylphenylketone thioether) (+)-E2-33 (15.6mg, 19.9 μmol, 1 equiv) in acetonitrile (150 μL) at 23° C., and thereaction was placed under a balloon of oxygen. The clear solutionimmediately turned orange. After 1 h, the reaction was diluted withdichloromethane (3 mL) and washed with saturated aqueous ammoniumchloride solution (3 mL). The aqueous layer was extracted with ethylacetate (3×2 mL), and the combined organic layers were dried over sodiumsulfate, were filtered, and were concentrated under reduced pressure.The orange residue was purified by flash column chromatography on silicagel (eluent: 3% acetone in dichloromethane) to afford the 3-propylpentacyclic epidithiodiketopiperazine E2-35 (5.9 mg, 57.4%) as a whitesolid. ¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.80 (d, J=7.0, 2H,SO₂Ph-o-H), 7.53 (app-t, J=7.0, 1H, SO₂Ph-p-H), 7.46-7.37 (m, 1H, C₈H),7.46-7.37 (m, 2H, SO₂Ph-m-H), 7.29, (app-dt, J=1.1, 7.7, 1H, C₇H), 7.16(app-t, J=7.7, 1H, C₆H), 7.12 (d, J=7.6, 1H, C₅H), 6.09 (s, 1H, C₂H),3.19 (d, J=15.2, 1H, C₁₂H_(a)), 2.98 (s, 2H, C₁₈H), 2.57 (d, J=15.2, 1H,C₁₂H_(b)), 1.87 (S, 3H, C₁₇H), 1.43-1.30 (m, 1H, CH₂CH₂CH₃), 1.22-1.04(m, 2H, CH₂CH₂CH₃), 0.77-0.68 (m, 2H, CH₂CH₂CH₃). ¹³C NMR (125.8 MHz,CDCl₃, 20° C.): δ 165.9 (C₁₃), 161.6 (C₁₆), 142.1 (C₉), 139.8(SO₂Ph-ipso-C), 137.6 (C₄), 133.4 (SO₂Ph-p-C), 129.3 (C₇), 129.2(SO₂Ph-m-C), 127.4 (SO₂Ph-o-C), 125.9 (C₆), 123.6 (C₅), 118.4 (C₈), 83.7(C₂), 73.7 (C₁₁), 73.5 (C₁₅), 55.9 (C₃), 41.8 (C₁₂), 40.0 (CH₂CH₂CH₃),27.7 (C₁₈), 18.3 (CH₂CH₂CH₃), 18.0 (C₁₇), 14.3 (CH₂CH₂CH₃). FTIR (thinfilm) cm⁻¹: 2960 (w), 1713 (s), 1688 (s), 1478 (w), 1460 (w), 1341 (m),1172 (m), 1092 (w). HRMS (ESI) (m/z): calc'd for C₂₄H₂₅NaN₃O₄S₃ [M+Na]⁺:538.0899, found: 538.0923. TLC (1% acetone in dichloromethane), Rf: 0.21(UV, CAM).

The compound was prepared using similar procedures as described above.¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.52 (d, J=7.7, 1H), 7.17 (app-t,J=7.7, 1H), 7.05 (app-t, J=7.7, 2H), 6.73 (s, 1H), 3.82 (app-dt, J=3.5,14.4, 1H), 3.48 (d, J=15.1, 1H), 3.30 (app-dt, J=3.5, 14.1, 1H), 2.99(s, 3H), 2.82 (d, J=14.8, 1H), 1.93 (s, 3H), 1.25-1.13 (m, 1H), 0.98(app-dt, J=3.9, 14.4, 1H), 0.05 (s, 9H).

The compound was prepared using similar procedures as described above.¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.93 (d, J=7.5, 2H), 7.54 (d, J=8.3,1H), 7.51 (t, J=7.3, 1H), 7.40 (app-t, J=7.9, 2H), 7.34 (d, J=7.5, 1H),7.29 (app-t, J=7.7, 1H), 7.11 (app-t, J=7.5, 1H), 6.23 (s, 1H), 4.97 (d,J=5.0, 1H), 4.74 (d, J=5.1, 1H), 4.61 (dd, J=6.2, 9.0, 1H), 4.39-4.23(m, 3H), 3.39 (dd, J=6.1, 14.3, 1H), 2.99 (dd, J=9.0, 14.3, 1H).

The compound was prepared using similar procedures as described above.¹H NMR (500 MHz, CDCl₃, 20° C.): δ 7.85 (d, J=7.2, 2H), 7.68 (d, J=6.4,1H), 7.57-7.51 (m, 1H), 7.49-7.43 (m, 2H), 7.22-7.12 (m, 2H), 7.01 (d,J=7.7, 1H), 6.83 (s, 1H), 3.79 (d, J=11.6, 1H), 3.70 (d, J=11.7, 1H),3.53 (d, J=15.0, 1H), 3.36 (s, 3H), 3.07 (s, 3H), 2.96 (d, J=15.2, 1H).

Compound N-1:

Compound N-1 was prepared as illustrated above. After purification onsilica column, compound N-1 was used for next step. ¹H NMR (400 MHz,CDCl₃, 20° C.): δ 6.04 (br-s, 1H), 4.95 (br-s, 1H), 4.30 (m, 2H),3.59-3.52 (m, 8H), 3.44 (m, 2H), 3.30 (br-s, 2H), 2.21 (m, 2H), 1.73 (m,4H), 1.42 (s, 9H).

Compound N-2 was prepared using similar procedure as those described forother EPT compounds. Reaction of N-2 with N-1 provided compound N-3 in64% yield. It is understood that based on the chemistry describedherein, a person having ordinary skill in the art can readily prepare anantibody-drug conjugate from a provided compound, for example, N-2. Itis also understood that compound N-3 is readily de-protected to providea free amino group, which can be used for conjugation to provideantibody-drug conjugates. For example, a hydroxyl group or an aminogroup can be linked to L and/or M through reaction with an activatedcarbonyl group to form an ester, carbonate, amide or carbamate.

¹H NMR (400 MHz, CDCl₃, 20° C.): δ 7.85 (m, 4H), 7.67 (d, J=6.9, 2H),7.56 (m, 2H), 7.48 (app-t, J=7.9, 4H), 7.23-7.16 (m, 4H), 7.06 (d,J=8.6, 2H), 6.83 (s, 2H), 4.12 (dd, J=4.2, 12.4, 2H), 3.84 (dd, J=4.9,12.4, 2H), 3.57 (d, J=15.2, 2H), 3.03 (s, 6H), 2.96 (d, J=15.1, 2H),2.73 (t, J=7.8, 2H).

¹H NMR (400 MHz, CDCl₃, 20° C.): δ 7.86 (m, 4H), 7.64 (d, J=7.2, 2H),7.57 (m, 2H), 7.48 (app-t, J=8.0, 4H), 7.24-7.14 (m, 4H), 7.03 (d,J=8.1, 2H), 6.84 (s, 2H), 6.04 (br-s, 2H), 4.98 (br-s, 2H), 4.50 (s,4H), 4.16 (m, 4H), 3.59-3.52 (m, 18H), 3.44 (m, 4H), 3.30 (m, 4H), 3.06(s, 6H), 2.92 (d, J=15.2, 2H), 2.21 (m, 4H), 1.71 (m, 8H), 1.42 (s,18H).

In some embodiments, a compound such as N-2 or deprotected N3 has morethan one functional group that can be used for conjugation. In someembodiments, a drug unit D is linked to two or more L and/or M units.

In some embodiments, a drug unit D is linked to one L and/or M unit. Itis understood that single linkage to L can be achieved through, forexample, selective protection/de-protection. An example is illustratedbelow:

In the example above, N-4 is selectively deprotected, or N-5 ismonoacetylated, to provide N-6. N-6 can be conjugated, for example, byreaction with activated carbonyl group, to form N-7. Alternatively, N-6can be further modified to provide N-8, which can be subsequentlyconjugated to provide N-9. In an exemplary N-9, s is 1, L is

wherein the drug unit is connected to L through the —C(O)—O— group, andM is a chimeric IgG1 antibody cAC10 specific for human CD30. In someembodiments, the acetyl group of N-8 was removed, either throughalternative synthesis pathway or through deprotection, providing N-8′.For example, in some embodiments, N-8′ was prepared through directmono-acylation of N-5. After removal of Boc, N-8′ can be conjugatedbased on known chemistry to provide N-9′, wherein the drug unit is N-8′.In an exemplary N-9′, s is 1, L is

wherein the drug unit is connected to L through the —C(O)—O— group, andM is a chimeric IgG1 antibody cAC10 specific for human CD30.

¹H NMR (400 MHz, CD₂Cl₂, 20° C.): δ 8.08 (d, J=7.4, 4H), 7.55 (t, J=7.4,2H), 7.45 (t, J=8.0, 4H), 7.34-7.23 (m, 20H), 7.08 (t, J=7.4, 2H), 7.02(d, J=7.4, 12H), 6.71 (m, 4H), 6.58 (t, J=7.4, 2H), 4.61 (d, J=11.4,2H), 3.98 (d, J=11.4, 2H), 3.21 (d. J=14.4, 2H), 2.80 (d, J=14.5, 2H),2.61-2.54 (m, 8H), 1.69 (s, 6H), 1.18 (d, J=7.0, 6H), 1.11 (d, J=7.0,6H).

¹H NMR (400 MHz, CDCl₃, 20° C.): δ 7.87 (m, 4H), 7.64 (d, J=8.3, 2H),7.56 (m, 2H), 7.47 (app-t, J=8.0, 4H), 7.24-7.16 (m, 4H), 7.03 (d,J=8.1, 2H), 6.85 (s, 2H), 4.42 (m, 4H), 3.58 (d, J=15.2, 2H), 3.04 (s,6H), 2.92 (d, J=15.2, 2H), 2.12 (s, 6H).

¹H NMR (400 MHz, CDCl₃, 20° C.): δ 7.85 (m, 4H), 7.67 (d, J=6.9, 2H),7.56 (m, 2H), 7.48 (app-t, J=7.9, 4H), 7.23-7.16 (m, 4H), 7.06 (d,J=8.6, 2H), 6.83 (s, 2H), 4.12 (dd, J=4.2, 12.4, 2H), 3.84 (dd, J=4.9,12.4, 2H), 3.57 (d, J=15.2, 2H), 3.03 (s, 6H), 2.96 (d, J=15.1, 2H),2.73 (t, J=7.8, 2H).

Compound N-8′: ¹H NMR (400 MHz, CDCl₃, 20° C.): δ 7.86 (m, 4H), 7.65(app-t, J=7.0, 2H), 7.56 (m, 2H), 7.47 (m, 4H), 7.23-7.15 (m, 4H), 7.04(m, 2H), 6.85 (s, 1H), 6.81 (s, 1H), 6.02 (br-s, 1H), 4.97 (br-s, 1H),4.50 (m, 2H), 4.16-4.09 (m, 3H), 3.84 (dd, J=5.0, 12.5, 1H), 3.58-3.52(m, 10H), 3.44 (m, 2H), 3.29 (m, 2H), 3.06 (s, 3H), 3.03 (s, 3H),2.96-2.91 (m, 2H), 2.77 (t, J=7.8, 1H), 2.20 (m, 2H), 1.70 (m, 4H), 1.42(s, 9H).

Among other things, the present invention provides ready syntheticaccess to ETP or thiodiketopiperazine compounds or derivatives andanalogs thereof; the present invention also provides detailed knowledgeof sites that can be modified without loss of activity, and that ETP orthiodiketopiperazine compounds or derivatives and analogs thereof do nothave hemolytic activity. The present invention, among other things,recognizes that ETP or thiodiketopiperazine compounds or derivatives andanalogs thereof can be used as drug units for ligand-drug conjugates. Insome embodiments, the present invention provides ligand-drug conjugates(“conjugate compound”). In some embodiments, a ligand-drug conjugate isan antibody-drug conjugate. In some embodiments, a provided compound hasthe structure of formula II. In some embodiments, a provided compoundhas the structure of formula II-a or II-b. In some embodiments, acompound of formula II-a or II-b is an ADC. Chemistry for conjugatingETP or thiodiketopiperazine compounds or derivatives and analogs thereofto ligands such as antibodies are widely known and practiced in the art,including those described in the present specification. Combining withmethods provided herein for synthesizing and modifying ETP orthiodiketopiperazine compounds (or derivatives or analogs thereof), awide array of ADCs can be prepared, including those having the structureof formula II-a or II-b. Methods for assaying a provided compound,including a provided ADC, are also widely known and practiced in theart. In some embodiments, a provided compound is tested in cancer celllines in culture and/or in in vivo tumor models, for example, thosedescribed above, and also in D. Greiner, T. Bonaldi, R. Eskeland, E.Roemer and A. Imhof, Nat. Chem. Biol., 2005, 1, 143; C. R. Isham, J. D.Tibodeau, W. Jin, R. Xu, M. M. Timm and K. C. Bible, Blood, 2007, 109,2579; Y. Chen, H. Guo, Z. Du, X.-Z. Liu, Y. Che and X. Ye, Cell Prolif,2009, 42, 838; Y.-M. Lee, J.-H. Lim, H. Yoon, Y.-S. Chun and J.-W. Park,Hepatology, 2011, 53, 171; F. Liu, Q. Liu, D. Yang, W. B. Bollag, K.Robertson, P. Wu and K. Liu, Cancer Res., 2011, 71, 6807; N. Zhang, Y.Chen, R. Jiang, E. Li, X. Chen, Z. Xi, Y. Guo, X. Liu, Y. Zhou, Y. Cheand X. Jiang, Autophagy, 2011, 7, 598; H. Chaib, A. Nebbioso, T. Prebet,R. Castellano, S. Garbit, A. Restouin, N. Vey, L. Altucci and Y.Collette, Leukemia, 2012, 26, 662; C. R. Isham, J. D. Tibodeau, A. R.Bossou, J. R. Merchan and K. C. Bible, Br. J. Cancer, 2012, 106, 314; M.Takahashi, Y. Takemoto, T. Shimazu, H. Kawasaki, M. Tachibana, Y.Shinkai, M. Takagi, K. Shin-ya, Y. Igarashi, A. Ito and M. Yoshida, J.Antiobiot., 2012, 65, 263; Y. Teng, K. Iuchi, E. Iwasa, S. Fujishiro, Y.Hamashima, K. Dodo and M. Sodeoka, Bioog. Med. Chem. Lett., 2010, 20,5085; M. Sodeoka, K. Dodo, Y. Teng, K. Iuchi, Y. Hamashima, E. Iwasa andS. Fujishiro, Pure Appl. Chem., 2012, 84, 1369; J. D. Tibodeau, L. M.Benson, C. R. Isham, W. G. Owen and K. C. Bible, Antiox. Redox Signal.,2009, 11, 1097: and Beverly A. Teicher (Editor), Tumor Models in CancerResearch (Cancer Drug Discovery and Development), 2^(nd) Ed, HumanaPress, 2011 (Publication date, Dec. 2, 2010). In some embodiments, aprovided compound demonstrates activities and efficacy for treatingcancer.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

1. A compound having the structure of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein:

is a single bond or a double bond, as valency permits; R¹ is R, —C(O)R,—C(O)N(R)₂, —S(O)R, —S(O)₂R, —S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂; each Ris independently hydrogen or an optionally substituted group selectedfrom C₁₋₂₀ aliphatic, C₁₋₂₀ heteroalkyl, phenyl, a 3-7 memberedsaturated or partially unsaturated carbocyclic ring, an 8-14 memberedbicyclic or polycyclic saturated, partially unsaturated or aryl ring, a5-6 membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, a 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, a7-14 membered bicyclic or polycyclic saturated or partially unsaturatedheterocyclic ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic or polycyclicheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; or: two R groups are optionally takentogether with their intervening atoms to form an optionally substituted3-14 membered, saturated, partially unsaturated, or aryl ring having, inaddition to the intervening atoms, 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; R² is R, —[C(R)₂]_(q)—OR,—[C(R)₂]_(q)—N(R)₂, —[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃,—[C(R)₂]_(q)—OC(O)R, —[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,—[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or R¹ and R² aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered heterocyclic ring having, in addition to thenitrogen atom to which R¹ is attached, 0-2 heteroatoms independentlyselected from oxygen, nitrogen or sulfur; each q is independently 0, 1,2, 3, or 4; R³ is an electron-withdrawing group; R⁴ is absent when

is a double bond or is R or halogen; R⁵ is absent when

is a double bond or is hydrogen or an optionally substituted C₁₋₆aliphatic group; each of R⁶ and R^(6′) is independently R, halogen, —CN,—NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,—C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, or—OSi(R)₃; or R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;n is 0, 1, 2, 3, or 4; each R⁷ is independently R, halogen, —CN, —NO₂,—OR, —OSi(R)₃, —SR, —N(R)₂, —S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂,—N(R)S(O)₂R, —P(R)₂, —P(OR)₂, —P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂,—B(R)₂, —B(OR)₂, or —Si(R)₃; or: two R⁷ are taken together with theirintervening atoms to form an optionally substituted 4-7 membered ringhaving 0-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur; R⁸ is —(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂;and R⁹ is —(S)_(p)—R^(y) wherein p is 1-3 such that m+p is 2-4 and R^(y)is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or—S(O)₂N(R)₂; or R⁸ and R⁹ are taken together to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—S)_(p)—, —(S)_(m)—C(O)—(S)_(p)—,—(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—.
 2. A compound having the structure of formulaI-b:

or a pharmaceutically acceptable salt thereof, wherein: each R¹ isindependently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R, —S(O)₂OR,—C(R)₂OR, or —S(O)₂N(R)₂; each R is independently hydrogen or anoptionally substituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated,partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups are optionally taken together with their intervening atomsto form an optionally substituted 3-14 membered, saturated, partiallyunsaturated, or aryl ring having, in addition to the intervening atoms,0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,—[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,—[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,—[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or R¹ and R² aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered heterocyclic ring having, in addition to thenitrogen atom to which R¹ is attached, 0-2 heteroatoms independentlyselected from oxygen, nitrogen or sulfur; each q is independently 0, 1,2, 3, or 4; each R³ is independently an electron-withdrawing group; eachR⁵ is independently hydrogen or an optionally substituted C₁₋₆ aliphaticgroup; each of R⁶ and R^(6′) is independently R, halogen, —CN, —NO₂,—OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR,—C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, or—OSi(R)₃; or R⁶ and R^(6′) are taken together to form ═O, ═C(R)₂ or ═NR;each n is independently 0, 1, 2, 3, or 4; each R⁷ is independently R,halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR, —N(R)₂, —S(O)₂R, —S(O)₂OR,—S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR,—N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, —P(R)₂, —P(OR)₂, —P(O)(R)₂,—P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂, —B(OR)₂, or —Si(R)₃; or two R⁷ aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; each R⁸ is independently—(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR, —C(O)R, —C(O)OR,—C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; and each R⁹ isindependently —(S)_(p)—R^(y) wherein p is 1-3 such that m+p is 2-4 andR^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R,or —S(O)₂N(R)₂; or R⁸ and R⁹ are taken together to form —S—,—(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p)—, —(S)_(m)—C(O)(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—.
 3. A compound having the structure of formulaII:MLD)s]_(t)   II or a pharmaceutically acceptable salt thereof,wherein: M is a cell-specific ligand unit; each L is independently alinker unit; each D independently has the structure of formula I-c orI-d,

or a pharmaceutically acceptable salt thereof, wherein: each

is independently a single bond or a double bond, as valency permits;each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R,—S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂; each R is independently hydrogen oran optionally substituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated,partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups are optionally taken together with their intervening atomsto form an optionally substituted 3-14 membered, saturated, partiallyunsaturated, or aryl ring having, in addition to the intervening atoms,0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,—[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,—[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,—[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or R¹ and R² aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered heterocyclic ring having, in addition to thenitrogen atom to which R¹ is attached, 0-2 heteroatoms independentlyselected from oxygen, nitrogen or sulfur; each q is independently 0, 1,2, 3, or 4; each R³ is independently R or an electron-withdrawing group;each R⁴ is independently absent when

is a double bond or is independently R or halogen; each R⁵ isindependently absent when

is a double bond or is independently hydrogen or an optionallysubstituted C₁₋₆ aliphatic group; each of R⁶ and R^(6′) is independentlyR, halogen, —CN, —NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂,—N(R)S(O)₂R, or —OSi(R)₃; or R⁶ and R^(6′) are taken together to form═O, ═C(R)₂ or ═NR; each n is independently 0, 1, 2, 3, or 4; each R⁷ isindependently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR, —N(R)₂,—S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂,—C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, —P(R)₂, —P(OR)₂,—P(O)(R)₂, —P(O)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂, —B(OR)₂, or —Si(R)₃; ortwo R⁷ are taken together with their intervening atoms to form anoptionally substituted 4-7 membered ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; each R⁸ isindependently —(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂;and each R⁹ is independently —(S)_(p)—R^(y) wherein p is 1-3 such thatm+p is 2-4 and R^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R,—S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or R⁸ and R⁹ are taken together to form—S—, —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—(S)_(p),—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—, or—(S)_(m)—S(O)₂—(S)_(p)—; s is 1-10; and t is 1-10. 4-6. (canceled)
 7. Acompound having the structure of formula III:H-LD)s   III or a pharmaceutically acceptable salt thereof, wherein: Lis a linker unit; each D independently has the structure of formula I-cor I-d,

or a pharmaceutically acceptable salt thereof, wherein: each

is independently a single bond or a double bond, as valency permits;each R¹ is independently R, —C(O)R, —C(O)N(R)₂, —S(O)R, —S(O)₂R,—S(O)₂OR, —C(R)₂OR, or —S(O)₂N(R)₂; each R is independently hydrogen oran optionally substituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturatedcarbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated,partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, a 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups are optionally taken together with their intervening atomsto form an optionally substituted 3-14 membered, saturated, partiallyunsaturated, or aryl ring having, in addition to the intervening atoms,0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R² is independently R, —[C(R)₂]_(q)—OR, —[C(R)₂]_(q)—N(R)₂,—[C(R)₂]_(q)—SR, —[C(R)₂]_(q)—OSi(R)₃, —[C(R)₂]_(q)—OC(O)R,—[C(R)₂]_(q)—OC(O)OR, —[C(R)₂]_(q)—OC(O)N(R)₂,—[C(R)₂]_(q)—OC(O)N(R)—SO₂R or —[C(R)₂]_(q)—OP(OR)₂; or R¹ and R² aretaken together with their intervening atoms to form an optionallysubstituted 4-7 membered heterocyclic ring having, in addition to thenitrogen atom to which R¹ is attached, 0-2 heteroatoms independentlyselected from oxygen, nitrogen or sulfur; each q is independently 0, 1,2, 3, or 4; each R^(3′) is independently R or an electron-withdrawinggroup; each R⁴ is independently absent when

is a double bond or is independently R or halogen; each R⁵ isindependently absent when

is a double bond or is independently hydrogen or an optionallysubstituted C-6 aliphatic group; each of R⁶ and R^(6′) is independentlyR, halogen, —CN, —NO₂, —OR, —SR, —N(R)₂, —S(O)₂R, —S(O)₂N(R)₂, —S(O)R,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂,—N(R)S(O)₂R, or —OSi(R)₃; or R⁶ and R^(6′) are taken together to form═O, ═C(R)₂ or ═NR; each n is independently 0, 1, 2, 3, or 4; each R⁷ isindependently R, halogen, —CN, —NO₂, —OR, —OSi(R)₃, —SR, —N(R)₂,—S(O)₂R, —S(O)₂OR, —S(O)₂N(R)₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R)₂,—C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R)₂, —N(R)S(O)₂R, —P(R)₂, —P(OR)₂,—P(O)(R)₂, —P(OP)(OR)₂, —P(O)[N(R)₂]₂, —B(R)₂, —B(OR)₂, or —Si(R)₃; ortwo R⁷ are taken together with their intervening atoms to form anoptionally substituted 4-7 membered ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, each R⁸ isindependently —(S)_(m)—R^(x) wherein m is 1-3 and R^(x) is R, —SR,—C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R, —S(O)R, —S(O)₂R, or —S(O)₂N(R)₂;and each R⁹ is independently —(S)_(p)—R^(y) wherein p is 1-3 such thatm+p is 2-4 and R^(y) is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R)₂, —C(S)R,—S(O)R, —S(O)₂R, or —S(O)₂N(R)₂; or R⁸ and R⁹ are taken together to form—S—, —(S)_(m)—[C(R)₂]_(q)—(S)_(p)—, —(S)_(m)—S)_(p)—,—(S)_(m)—C(O)—(S)_(p)—, —(S)_(m)—C(S)—(S)_(p)—, —(S)_(m)—S(O)—(S)_(p)—,or —(S)_(m)—S(O)₂—(S)_(p)—; and s is 1-10. 8-15. (canceled)
 16. Apharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 17. A method for treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of a compound of claim 1, or a pharmaceutically acceptable saltthereof.
 18. A method for treating an autoimmune disease in a subject,comprising administering to the subject a therapeutically effectiveamount of a compound of claim 1, or a pharmaceutically acceptable saltthereof.
 19. A method for generating reactive oxygen species, comprisingproviding a compound of claim 1, or a pharmaceutically acceptable saltthereof.
 20. A method for inhibiting a protein, comprising providing acompound of claim 1, or a pharmaceutically acceptable salt thereof. 21.A method for disrupting structures of proteins containing a Zn²⁺,comprising providing a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 22. A method for inducing apoptosis, comprisingproviding a compound of claim 1, or a pharmaceutically acceptable saltthereof.
 23. A method for killing or inhibiting proliferation of cellscomprising treating the cells with an amount of a compound of claim 1,or a pharmaceutically acceptable salt thereof, being effective to killor inhibit proliferation of the cells. 24-31. (canceled)
 32. Apharmaceutical composition comprising a compound of claim 2, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 33. A method for treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of a compound of claim 2, or a pharmaceutically acceptable saltthereof.
 34. A method for treating an autoimmune disease in a subject,comprising administering to the subject a therapeutically effectiveamount of a compound of claim 2, or a pharmaceutically acceptable saltthereof.
 35. A method for generating reactive oxygen species, comprisingproviding a compound of claim 2, or a pharmaceutically acceptable saltthereof.
 36. A method for inhibiting a protein, comprising providing acompound of claim 2, or a pharmaceutically acceptable salt thereof. 37.A method for disrupting structures of proteins containing a Zn²⁺,comprising providing a compound of claim 2, or a pharmaceuticallyacceptable salt thereof.
 38. A method for inducing apoptosis, comprisingproviding a compound of claim 2, or a pharmaceutically acceptable saltthereof.
 39. A method for killing or inhibiting proliferation of cellscomprising treating the cells with an amount of a compound of claim 2,or a pharmaceutically acceptable salt thereof, being effective to killor inhibit proliferation of the cells.